CN110227075A - Purposes of the carnosol as inflammation corpusculum inhibitor - Google Patents

Abstract

The application discloses purposes of the carnosol as inflammation corpusculum inhibitor.Carnosol has inhibiting effect to various inflammation corpusculums, is the inflammation corpusculum inhibitor of wide spectrum, for developing the drug for treating acquired inflammatory disease and autoimmunity disease.

Description

Purposes of the carnosol as inflammation corpusculum inhibitor

Technical field

This application involves drug screening field, especially but not limited to being related to use of the carnosol as inflammation corpusculum inhibitor On the way.

Background technique

Inflammation corpusculum is a kind of intracellular large-scale multiprotein complex, by receptor protein, apoptosis associated speck-like protein Before (apoptosis-associated speck-like protein, ASC) and caspases specific proteases 1 Body (pro-caspase-1) composition, inflammation corpusculum by pattern recognition receptors (pattern recognition receptor, PRR pathogenic microorganism and endogenous danger signal, i.e. pathogen-associated molecular pattern (pathogenassociated) are identified Molecular pattern, PAMP) and dangerous associated molecular pattern (damage associated molecular Pattern, DAMP), recruit and activate caspase-1, shear IL-1 β and IL-18 precursor, generate corresponding mature cell because Son participates in body to the removing of pathogen and the generation of adaptive immune response to promote Apoptosis.Presently found inflammation Disease corpusculum is named as nucleotide combination oligomerization domain receptor protein 1 according to the difference of receptor protein respectively (nucleotide-binding oligomerization domain-like receptor protein 1, NLRP1), core 3 (nucleotide-binding oligomerization domain-like of thuja acid combination oligomerization domain receptor protein Receptor protein 3, NLRP3), nucleotide combination oligomerization domain receptor CARD domain protein 4 (nucleotide-binding oligomerization domain-like receptor CARD-domain Containing protein 4, NLRC4), melanoma lack the factor 2 (Absent in melanoma 2, AIM2) inflammation Corpusculum.

Important component of the inflammation corpusculum as inherent immunity, abnormal activation and functional disturbance and a variety of acquired inflammation The pathologic process of property disease and autoimmunity disease is closely related.It has now been found that ulcerative colitis, Alzheimer disease, 2 Patients with type Ⅰ DM, atherosclerosis, gout and nonalcoholic fatty liver disease etc. all with the close phase of the abnormal activation of inflammation corpusculum It closes.But there is not the clinical application of related targeted drug in terms for the treatment of acquired inflammatory disease and autoimmunity disease at present, because This exploitation inflammation corpusculum inhibitor, inhibits the abnormal activation of inflammation corpusculum, is beneficial to the treatment of above-mentioned disease and provides for it New therapy approach.

Summary of the invention

It is the general introduction to the theme being described in detail herein below.This general introduction is not the protection model in order to limit claim It encloses.

In recent years, some small molecule compounds have shown that the potential inhibiting effect to the activation of NLRP3 inflammatory body, such as 3,4- methylene-dioxies-nitrostyrolene, glibenclamide, parithenolide, sulforaphen, isoliquiritigenin, MCC950, beta-hydroxy Butyrate, Flufenamic acid and mefenamic acid acid etc., but have the compound of inhibiting effect less various inflammation corpusculums.Salvia japonica Phenol (Carnosol), CAS 5957-80-2, molecular formula C20H26O4, relative molecular mass 330.424g/mol.Modern study Show carnosol its have the effects that it is anti-inflammatory, antitumor.Document report, carnosol, which passes through, inhibits NF- κ B and MAPK etc., Reduce the release of proinflammatory cytokines such as tumor necrosis factor (TNF-α), but the potential mechanism of its anti-inflammatory effect and direct target Or it is unknown.And inflammation corpusculum plays the effect of core in inflammation disease, therefore, studies carnosol to inflammation corpusculum Regulating and controlling effect and its effect in treatment diseases associated with inflammation are of great significance.Carnosol (Camosol) derives from lip Shape section Rosmarinus plant rosemary, CAS 5957-80-2, molecular formula C20H26O4, relative molecular mass 330.424g/ Mol, colourless crystallization are dissolved in DMSO, have the multiple efficacies such as anti-radiation, anti-oxidant, anti-inflammatory, anticancer.

Carnosol

Specifically, the application provides purposes of the carnosol as inflammation corpusculum activation inhibitor.

In this application, purposes of the carnosol in the drug of preparation treatment inflammation corpusculum abnormal activation related disease.

In this application, the inflammation corpusculum can be selected from the hot protein structure domain 3 of oligomerization domain sample receptor family (NLRP3) inflammation corpusculum, oligomerization domain sample receptor family caspase activation raise structural domain 4 (NLRC4), melanin Tumor lacks any one of the factor 2 (AIM2) or more.

In this application, carnosol can inhibit the expression of one or both of caspase-1 and IL-1 β.

In this application, carnosol can inhibit infectious shock.

In this application, carnosol can reduce death caused by gram positive bacterial infection.

In this application, the related disease can be selected from ulcerative colitis, Alzheimer disease, diabetes B, move Any one of pulse atherosclerosis, gout and nonalcoholic fatty liver disease or more.

In this application, the administration dosage of carnosol can be 20mg/kg-100mg/kg.

In this application, the method for application of carnosol can be oral or injection.

Applicants have discovered that: 1. carnosols to inflammation corpusculum activate it is inhibited, can inhibit NLRP3, NLRC4, The activation of the inflammations corpusculum such as AIM2, is the inflammation corpusculum inhibitor of wide spectrum.

2. acquired inflammatory disease and autoimmunity disease are related to inflammation corpusculum there are many at present, such as ulcerative colitis Inflammation, Alzheimer disease, diabetes B, atherosclerosis, gout and nonalcoholic fatty liver disease etc., but there is no needles To the targeted drug of such disease.The application has found that carnosol can inhibit the activation of inflammation corpusculum, and then for developing and can treat The drug of acquired inflammatory disease and autoimmunity disease.

3. the application discovery carnosol in the mouse sensible heat shock disease model that LPS is induced can significantly inhibit mouse sensible heat Disease of suffering a shock reacts and its caused death.

4. it has now been found that inhibiting the gene expression or suppression of caspase-1 in chemical colitis mouse model IL-1 β or IL-18 processed can alleviate colitis.Britain " Gut " gastrointestinal disease magazine in 2010 delivers the colitis of DSS induction Article the study found that the secretion for knocking out the IL-1 β of the macrophage of NLRP3 or ASC, caspase-1 encoding gene is significant Decline, the NLRP3 after orally administration DSS intervention^-/-Mouse shows the colitis lighter compared with wild-type mice and lower Inflammatory factor expression is horizontal, and reaches after Drug inhibition colitis mice caspase-1p20 and NLRP3^-/-Mouse is similar glutinous Film protective effect, this shows that DSS is by NLRP3 inflammation corpusculum Pathway Activation inducing colitis disease；Mouse is given through intraperitoneal injection After medicine carnosol, the mouse colitis symptom of chemically DSS induction can be significantly improved.Document report, MCC950 is in nanomole Rank can completely inhibit the activation of NLRP3 inflammation corpusculum, and can in-vitro simulated NLRP3^-/-Mouse.The application is in MCC950 mould Quasi- NLRP3^-/-Orally administration DSS in mouse, intraperitoneal injection carnosol on this basis, to prove carnosol By inhibiting NLRP3 activation to play the role for the treatment of murine colonic inflammation.

5. it is thin that non-alcohol fatty liver (NAFLD) can increase cardiovascular event, cancer, the death rate of cirrhosis and liver Born of the same parents' cancer, and NLRP3 inflammation corpusculum play the role of in non-alcohol fatty liver it is vital.The application has found Salvia japonica Phenol can significantly reduce the hepatic fibrosis in mice degree in mouse NASH model, be conducive to face by inhibiting NLRP3 inflammation corpusculum Bed exploitation can treat the targeted drug of NASH.

Other features and advantage will illustrate in the following description, also, partly become from specification It obtains it is clear that being understood and implementing the application.The purpose of the application and other advantages can be by specifications, right Specifically noted structure is achieved and obtained in claim and attached drawing.

Detailed description of the invention

Attached drawing is used to provide to further understand technical scheme, and constitutes part of specification, with this The embodiment of application is used to explain the technical solution of the application together, does not constitute the limitation to technical scheme.

The NLRP3 activation of Fig. 1 carnosol dose-dependent inhibition LPS+Nigerigin induction.Western blot detection NLRP3, ASC, pro-Caspase- in caspase-1 p20, the content of IL-1 β p17 and cell pyrolysis liquid in cell conditioned medium 1, pro-IL-1 β expresses (Figure 1A).Utilize caspase-1 activity in caspase-1 activity detection kit measurement cell conditioned medium (Figure 1B), ELISA kit detect the content (Fig. 1 C) of IL-1 β in cells and supernatant.

Fig. 2 carnosol significantly inhibits the activity of the inflammation corpusculum such as NLRP3, NLRC4, AIM2.Fig. 2A and Fig. 2 B is immune Blotting testing result, Fig. 2 C and Fig. 2 E are Caspase-1 contents in Caspase-1 activity detection kit detection cell conditioned medium As a result, Fig. 2 D and Fig. 2 F are the results of IL-1 β content in ELISA detection cell conditioned medium.

Fig. 3 carnosol treats the infectious shock of LPS induction, and significantly reduces dead caused by gram positive bacterial infection It dies.Fig. 3 A be using IL-1 β content in ELISA method detection mice serum as a result, Fig. 3 B be mouse peritoneal be administered 1 hour after, The survivorship curve of mouse after intraperitoneal injection LPS.

Fig. 4 carnosol treats the chmice acute ulcerative colitis of DSS induction.Fig. 4 A is mouse Colon picture, Fig. 4 B It is mouse Colon length, Fig. 4 C is the changes of weight of mouse.

The active treatment chmice acute ulcerative colitis that Fig. 5 carnosol passes through inhibition NLRP3 inflammation corpusculum.Fig. 5 A is Mouse Colon picture, Fig. 5 B are mouse Colon length, and Fig. 5 C is the changes of weight of mouse.

Fig. 6 carnosol treats non-alcoholic fatty type hepatitis.Fig. 6 A is to take mice serum detection ALT as a result, Fig. 6 B It is the result of mice serum detection AST.

Specific embodiment

For the purposes, technical schemes and advantages of the application are more clearly understood, below in conjunction with attached drawing to the application Embodiment be described in detail.It should be noted that in the absence of conflict, in the embodiment and embodiment in the application Feature can mutual any combination.

Drug:

Carnosol (Carnosol), MCC950 are purchased from TargetMol company；LPS is purchased from Sigma Co., USA.

Reagent, material:

DMEM culture medium, fetal calf serum (HyClone company, the U.S.)；(U.S. Gibco is public for Opti-MEM serum free medium Department)；Trypsase, dual anti-(Pen .- Strep solution) (stepping morning science and technology Beijing Co., Ltd)；Atriphos (ATP), Buddhist nun Day Leah rhzomorph (Nigericin), lipopolysaccharides (LPS), dimethyl sulfoxide (DMSO) (U.S.'s Sigma Aldrich)； NLRP3, ASC, Caspase-1, pro-Caspase-1, pro-IL1 β, GAPDH antibody (U.S. Cell Signaling Technology company)；CCK-8 kit (MCE)；Caspase-1 Activity Assay Kit (Promega company, the U.S.)； TNF-α, IL-1 β ELISA detection kit (it is Bioisystech Co., Ltd that Beijing, which reaches section)；5 × SDS protein electrophoresis loading is slow Fliud flushing, tri- color pre-dyed albumen Marker of PM2510 (Beijing Ding Guochang wins Bioisystech Co., Ltd)；Electrophoresis liquid, transfer liquid, 10 × TBST buffer, Coomassie brilliant blue (Beijing Bo Maide gene technology Co., Ltd)；Methanol, acetic acid (Beijing Chemical Plant)； RIPA lysate (the green skies Bioisystech Co., Ltd in Shanghai)；A, B developer solution, pvdf membrane (Millipore company, the U.S.).

Instrument:

Carbon dioxide incubator (Thermo Scientific company, the U.S.)；Microplate reader (Promega company, the U.S.)；It is low Warm supercentrifuge (Sigma Co., USA)；Electrophoresis tank, transfer groove, gel electrophoresis analysis system (Bio-Rad company, the U.S.)； Tissue culture plate shaker (German IKA company).

1. cellular level examine LPS respectively with atriphos (adenosine triphosphate, ATP), Buddhist nun day Leah rhzomorph (nigericin), silica (SiO2), Poly (I: C), Poly (dA: dT), Salmonella (Salmonella) under Co stituation, inhibiting effect of the carnosol to the inflammation corpusculum of various activation；Pam3csk4 stimulation with Transfect the inhibiting effect of the non-classical inflammation corpusculum of LPS activation.

2. carnosol can significantly inhibit the shock disease reaction of mouse sensible heat and its caused death.

3. the LPS of high dose can induce immunity of organism stress, cause the inflammatory factors such as TNF-α, IL-1 β secretion increase, The application has found that carnosol can significantly reduce the secretion increase of the inflammatory factors such as TNF-α caused by LPS, IL-1 β, reduces abdominal cavity Macrophage, neutrophil level.

4. the murine colonic inflammation that mouse after intraperitoneal injection carnosol, can significantly improve chemically DSS induction Shape, and in the NLRP3 of MCC950 simulation^-/-Orally administration DSS in mouse, then intraperitoneal injection carnosol, to prove mouse Tail grass phenol is by inhibiting the activation of NLRP3 inflammation corpusculum to play the role for the treatment of murine colonic inflammation.

5. in nonalcoholic fatty liver disease model, mouse peritoneal drug administration by injection carnosol can significantly reduce mouse Transaminase level, and it is obviously improved hepatic fibrosis in mice degree, illustrate that this carnosol can treat non-alcoholic fatty liver It is scorching.

Product prepares embodiment:

1. carnosol can significantly reduce IL-1 β caused by the activation of inflammation corpusculum under LPS and many factors Co stituation Secretion

A. the preparation of mouse primary bone marrow macrophage (BMDMs): taking 10 week old C57BL/6 female mices, separates humerus And femur, with DMEM culture medium (10% fetal calf serum) repeatedly bone marrow extraction cell and by 1: 4000 be added mouse colony stimulation because After sub (MCSF) breaks up 5 days, vitellophag simultaneously presses 9 × 105/ml kind plate (24 orifice plates, 0.5ml/well), adherent laggard overnight Row is tested in next step.

B.LPS stimulation: the mode for changing liquid is taken, BMDMs is added to the concentration (0.4ml/well) of 50ng/ml and is stimulated 4h。

C. dosing: the carnosol monomer for taking DMSO to dissolve is configured to 10 μM, then 2 times of dilutions with Opti-MEM culture medium At 5 μM, 2.5 μM of totally three concentration take the mode for changing liquid to be added in BMDMs (0.4ml/well), stimulate 1h.

The stimulation of d.NLRP3 stimulating factor: the mode of fluid infusion is taken, final concentration of 5mM ATP (0.02ml/ is separately added into Well 1h, 10 μM of nigericin (0.02ml/well)) is stimulated to stimulate 0.5h.The SiO2 (0.02ml/well) of 250 μ g/ml, The Poly (I: C) and Poly (dA: dT) (0.02ml/well) of 2 μ g/ml,

E. receive sample: cell receives sample, is directly added into 1 × RIPA Loading cracking (120 hole μ L/), shakes rapidly even, makes Loading covers all cells, and again in this way, 3 times repeatedly after 5mins, Loading is sucked in EP pipe.At cell conditioned medium sample Reason: room temperature 4000rpm is centrifuged 5mins, receives supernatant, and 1/4 volume trichloroacetic acid (TCA) is added, after 4 DEG C of standing 1h or more, 4 DEG C, 12000rpm is centrifuged 15mins；Supernatant is abandoned, ice acetone (500 hole μ L/) mixing of turning upside down is added, 4 DEG C, 12000rmp is centrifuged 15mins abandons supernatant, and 95 DEG C of metal baths volatilize acetone, and after acetone volatilization plus 1 × RIPA Loading, 35 μ L oscillation mixes, Water-bath is boiled, and is used as Supernatant samples after cooling.

F.Western blot: IL-1 β p17, caspase-1 p20 protein expression level in detection supernatant detect cell NLRP3, ASC, pro-Caspase-1 in lysate, pro-IL-1 β protein level.Cell conditioned medium protein sample is dense using 12% The SDS- polyacrylamide gel electrophoresis of degree, cell pyrolysis liquid sample are solidifying using the SDS- polyacrylamide of the concentration of 10% concentration Gel electrophoresis powers on, and sets constant voltage 80V, and voltage is changed to 135V after 45mins, when bromophenol blue reaches separation gel bottom, Terminate electrophoresis, transfer device is installed.After cell conditioned medium terminates electrophoresis, glue is cut out along the position maker 60kD, 60kD or more is used and examined Mas bright blue dye 30mins, destainer (water: methanol: acetic acid=5: 4: 1), until background blue take off it is transparent；The cathode of transfer groove 4 layers of filter paper are spread, the remaining gel of 60kD or less are laid on filter paper, and in glue upper berth pvdf membrane, 5 layers of filter paper are layered on the upper of film Side.Power on, set with constant current, 450mA, 3h.After transferring film, 5% defatted milk room temperature closes 1h, different primary antibodies (1: 1000) 4 DEG C of solution overnight incubations, pvdf membrane washes film 3 times through 1%TBS-T solution interval 5mins, then with the secondary antibodies of respective markers (1: 5000) solution carries out reaction 1h, abandons secondary antibody, is washed film 3 times with 1%TBS-T solution interval 5mins, and two kinds of developer solution A and B are tried It after agent mixes in equal volume, is added on film immediately, darkroom is developed using X-ray film tabletting Exposure mode.

G.ELISA: measuring the content of IL-1 β in cell supernatant, TNF-α, and operating procedure by specification carries out.? 450nm wavelength reads OD value, calculates sample concentration according to standard curve.

H.Caspase-1 determination of activity: it is operated according to Caspase-1 Activity Assay Kit specification.First room temperature Balance Caspase-1 buffer, Z-WEHD fluorescein substrate and MG-132 inhibitor, then in 10mL Z-WEHD- ammonia 60 μ L MG-132 (120 μM) inhibitor sufficient vortexes are added in base fluorescein substrate and, to being thoroughly mixed, add Caspase-1 buffer, sufficient vortex form final concentration of 60 μM of fluorescence detection mixture, for use.Every hole is added in 96 orifice plates The cell conditioned medium sample of 50 μ L, then every hole are added 30 μ L and prepare stand-by fluorescence detection mixture, the vibration of cell culture plate oscillator 1min is swung, room temperature, which is protected from light balance, combines sample sufficiently with fluorogenic substrate, tests and analyzes Caspase-1 fluorescence through microplate reader after 1h Value.

I. statistical method: data count soft using GraphPad Prism 6.0 (GraphPad, San Diego, CA) Part is handled, and group difference analysis selects One-way ANOVA method to carry out.P < 0.05 is considered that there are statistical differences.

2. carnosol can treat the infectious shock of LPS induction, and significantly reduces gram positive bacterial infection and cause Death:

A.C57BL/6 mouse, 7-8 weeks, female, was purchased from Beijing Si Beifu Bioisystech Co., Ltd, is randomly divided by 48 5 groups, respectively blank control group, model group, carnosol group (40mg/kg), carnosol+LPS group (40mg/kg), MCC950+LPS group (40mg/kg)；Blank control group, model group intraperitoneal injection 0.2ml contain the PBS solution of 10% Tween 80, Carnosol and MCC950 is injected intraperitoneally in remaining each group respectively；After 1 hour, except the PBS of blank control group intraperitoneal injection 0.2ml is molten Liquid, remaining each group inject the LPS of 0.2ml 20mg/kg respectively, and eyeball takes blood after 2 hours, takes peritoneal macrophage, takes 4 after blood DEG C centrifuging and taking serum, ELISA detect serum levels of inflammatory cytokines IL-1 β, TNF-α, peritoneal macrophage Flow cytometry abdominal cavity Macrophage and neutrophil leucocyte number, the results showed that carnosol can treat the infectious shock of LPS induction.

B. mouse survival rate curve: C57BL/6 mouse, 7-8 weeks, female 60 had purchased from Beijing Si Beifu biotechnology Limit company, in laboratory, adaptive feeding after a week, is randomly divided into 5 groups, respectively the model group, (administration of carnosol+LPS group Dosage is respectively 25mg/kg, 50mg/kg, 100mg/kg) and MCC950+LPS group (50mg/kg).Model group intraperitoneal injection 0.2ml contain 10% Tween 80 PBS solution, remaining group be injected intraperitoneally respectively different dosing dosage carnosol and MCC950；After 1 hour, the LPS of 0.2ml 20mg/kg is injected intraperitoneally in each group, observes and records mouse survival situation within every 3 hours, And draw mouse survival tracing analysis survival rate；As the result is shown in the mouse gram positive bacterial infection model of LPS induction, mouse Tail grass phenol can significantly reduce the death rate of mouse.

3. the chmice acute ulcerative colitis that carnosol treats DSS (Dextran sulfate sodium) induction.

C57BL/6 mouse, 8 weeks, female 36 was purchased from Beijing Si Beifu Bioisystech Co., Ltd, adapted in laboratory Property raising after a week, be randomly divided into 6 groups (n=6), respectively Normal group, carnosol (20mg/kg, 40mg/kg) group, DSS model group, DSS+ carnosol (20mg/kg, 40mg/kg) group.Normal group and carnosol group normally drink sterile steaming Distilled water 7 days, other each groups drank the sterile distilled water containing 2.5%DSS, except 0.2ml is injected intraperitoneally in Normal group and DSS group daily Outside PBS, 0.2ml carnosol (20mg/kg, 40mg/kg) is injected intraperitoneally in other each groups daily.Weighing record daily observes excrement Just, whether hematochezia, mouse activity condition, record mouse DAI scoring.Eyeball of mouse takes blood within 8th day, takes spleen to weigh, takes colon, Colon lengths are measured, rear end is rinsed well with PBS and cuts one section and be put into -80 DEG C of preservation, remaining colon is put into 10% neutrality good fortune It is fixed in your Malin's buffer.

4. the acute ulcer knot that carnosol mainly inhibits DSS to induce by inhibiting the activation of NLRP3 inflammation corpusculum Enteritis.

C57BL/6 mouse, 8 weeks, female 30 was purchased from Beijing Si Beifu Bioisystech Co., Ltd, adapted in laboratory Property raising after a week, be randomly divided into 5 groups (n=6), respectively Normal group, DSS model group, DSS+ carnosol (40mg/ Kg) group, DSS+MCC950 (40mg/kg) group, DSS+ carnosol (40mg/kg)+MCC950 (40mg/kg) group.Normal control Group and carnosol group normally drink sterile distilled water 7 days, other each groups drink the sterile distilled water containing 2.5%DSS, remove normal control Group and DSS group daily be injected intraperitoneally 0.2ml PBS outside, other each groups be injected intraperitoneally daily 0.2ml carnosol (20mg/kg, 40mg/kg), group containing MCC950 proposes the previous day intraperitoneal injection 0.2ml MCC950 (40mg/kg).Weighing record daily observes excrement Just, whether hematochezia, mouse activity condition, record mouse DAI scoring.Eyeball of mouse takes blood within 9th day, takes spleen to weigh, takes colon, Colon lengths are measured, rear end is rinsed well with PBS and cuts one section and be put into -80 DEG C of preservation, remaining colon is put into 10% neutrality good fortune It is fixed in your Malin's buffer.

5. carnosol is treated to nonalcoholic fatty liver disease.

C57BL/6 mouse, 8 weeks, female 60 was purchased from Beijing Si Beifu Bioisystech Co., Ltd, adapted in laboratory Property raising after a week, be randomly divided into 6 groups (n=10), be divided into and lack methionine and choline diet group (feeding MCD feed) and have no lack of Methionine and choline diet group (feeding MCS feed).MCD feed group and MCS feed ingredient are not divided into Normal group, carnosol (40mg/kg) group, MCC950 (40mg/kg) group.The daily gastric infusion 0.2ml PBS of control group mono- week, carnosol group and The daily gastric infusion of MCC950 group one week, was administered once, every two days after a week until 6 weeks.Eyeball of mouse takes blood after 6 weeks, takes 3500rpm/min centrifugation, surveys IL-1 β, TNF-α, the content of ALT, AST in serum；Mouse is dissected, hepatomegaly leaf is put into 10% neutrality It is fixed in formalin buffer, HE dyeing detection hepatocellular injury, tunnel dyeing detection hepatocellular apoptosis, remaining hepatic tissue It is put into -80 DEG C of preservations.

As a result:

1. then giving stimulating factor from figure 1 it appears that giving carnosol on the basis of LPS is pretreated Nigericin induces the activation of inflammation corpusculum, collects cell conditioned medium and cell pyrolysis liquid.Using Western blot in cell conditioned medium The small body associated protein of the inflammation such as NLRP3, ASC in caspase-1 p20 and maturation IL-1 β and cell pyrolysis liquid is detected, Carnosol is in the expression of the caspase-1 p20 and maturation IL-1 β in dose-dependent inhibition cell conditioned medium as the result is shown, But (Figure 1A) is not influenced on the expression of the small body associated proteins of inflammation such as NLRP3, ASC in cell pyrolysis liquid.Further pass through Caspase-1 Inflammasome Assay and ELISA detects caspase-1 activity and maturation in cell conditioned medium respectively The content of IL-1 β, carnosol can be in dose-dependent inhibition caspase-1 active (Figure 1B) and maturation IL-1 as the result is shown β (Fig. 1 C) secretion.

2. from figure 2 it can be seen that give carnosol on the basis of LPS is pretreated, then give ATP, Nigericin, SiO2, Poly (I: C), Poly (dA: dT), Salmonella etc. different stimulus induction inflammation corpusculums is living Change, collects cell conditioned medium and cell pyrolysis liquid.Using Western blot to caspase-1 p20 in cell conditioned medium and maturation IL-1 The small body associated proteins of inflammation such as NLRP3, ASC in β and cell pyrolysis liquid are detected, and carnosol significantly inhibits as the result is shown Various stimulus activate the expression of caspase-1 p20 and maturation IL-1 β in lower cell conditioned medium, but in cell pyrolysis liquid The expression of the small body associated protein of inflammation such as NLRP3, ASC do not influence (Fig. 2A, B).Further pass through Caspase-1 Inflammasome Assay and ELISA detect the content of caspase-1 activity and maturation IL-1 β in cell conditioned medium respectively, knot Fruit shows that carnosol significantly inhibits the activity (Fig. 2 C, E) and maturation IL-1 β (Fig. 2 D, F) secretion of caspase-1.

3. can be seen that in the mouse infection Shock Model that LPS is induced from Fig. 3 A, carnosol is to IL-1 in serum The secretion of β has significant inhibiting effect (P < 0.05).

4. as can be seen that carnosol significantly reduces death caused by gram positive bacterial infection from Fig. 3 B.

5. model group mouse Colon length becomes figure 4, it is seen that carnosol can improve mouse disease symptom Short, carnosol has apparent restitution to mouse Colon length.Compared with normal group, model group body weight is substantially reduced (P < 0.001) carnosol group is without significant change (P > 0.05)；And compared with model group, carnosol+DSS organizes weight loss situation It is obviously improved (P < 0.05).Compared with normal group, model group inflammatory factor IL-1 β significantly increases (P < 0.05), carnosol Group is without significant change；Compared with model group, carnosol+DSS, which organizes IL-1 β, significantly reduces (P < 0.05).

6. from fig. 5, it can be seen that model group mouse Colon length shortens, and mouse weight is obvious compared with Normal group It reduces, inflammatory factor IL-1 significantly increases (P < 0.05).Compared with model group, MCC950+DSS group, carnosol+DSS group, Carnosol+MCC950+DSS group mouse Colon length is obviously restored, and mouse weight obviously restores (P < 0.05), inflammatory factor IL-1 β significantly reduces (P < 0.05).Compared with carnosol+DSS group, MCC950+DSS group, carnosol+MCC950+DSS Group colon lengths, mouse weight, inflammatory factor IL-1 β are without significant difference.Illustrate that carnosol mainly passes through and inhibits NLRP3 inflammation Corpusculum activates to improve the acute ulcer colitis of DSS induction.

7. from fig. 5, it can be seen that ALT, AST are without significant difference between feeding MCS feed each group；Compared with only feeding MCS feed group, MCD group ALT, AST is only fed significantly to increase.Compared with the model group for only feeding MCD, gastric infusion mouse on the basis of feeding MCD feed Tail grass phenol and MCC950, ALT, AST of mouse are significantly reduced.Illustrate that carnosol has significant treatment to nonalcoholic fatty liver Effect.

It can be seen that in vitro experiment by the result of above embodiments, carnosol is able to suppress ATP, Ni Ni Various inflammation corpusculums caused by sub- rhzomorph, SiO2, Poly (I: C), Poly (dA: dT), Salmonella, transfection LPS etc. stimulate Activation.In vivo experiment, it has further been found that carnosol is substantially reduced dead caused by LPS infective shock symptom and infection It dies；Significantly improve the chmice acute ulcerative colitis symptom of chemically DSS induction.It can be seen that carnosol can be used as it is pre- Anti- or treatment and inflammation corpusculum abnormal activation related disease potential drug.

Although embodiment disclosed by the application is as above, the content only for ease of understanding the application and use Embodiment is not limited to the application.Technical staff in any the application fields, is taken off not departing from the application Under the premise of the spirit and scope of dew, any modification and variation, but the application can be carried out in the form and details of implementation Scope of patent protection, still should be subject to the scope of the claims as defined in the appended claims.

Claims (9)

1. purposes of the carnosol as inflammation corpusculum activation inhibitor.

2. purposes according to claim 1, wherein carnosol treats inflammation corpusculum abnormal activation related disease in preparation Drug in purposes.

3. purposes as claimed in claim 1 or 2, wherein the inflammation corpusculum is selected from oligomerization domain sample receptor family heat (NLRP3) inflammation of protein structure domain 3 corpusculum, oligomerization domain sample receptor family caspase activation raise structural domain 4 (NLRC4), melanoma lacks any one of the factor 2 (AIM2) or more.

4. purposes according to claim 2, wherein the related disease be selected from ulcerative colitis, Alzheimer disease, Any one of diabetes B, atherosclerosis, gout and nonalcoholic fatty liver disease or more.

5. purposes according to claim 1, wherein carnosol inhibits the expression in caspase-1 and IL-1 β.

6. purposes according to claim 1, wherein carnosol inhibits infectious shock.

7. purposes according to claim 1, wherein carnosol reduces death caused by gram positive bacterial infection.

8. according to claim 1, purposes described in any one of 2 and 4-7, wherein the administration dosage of carnosol is 20mg/ kg-100mg/kg。

9. according to claim 1, purposes described in any one of 2 and 4-7, wherein the method for application of carnosol be it is oral or Injection.