CN107823196A - Application of the carnosic acid in terms for the treatment of medicine for treating rheumatoid arthritis is prepared - Google Patents
Application of the carnosic acid in terms for the treatment of medicine for treating rheumatoid arthritis is prepared Download PDFInfo
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Abstract
The invention discloses application of the carnosic acid in terms of medicine for treating rheumatoid arthritis is treated.In vitro and in vivo experiment shows, carnosic acid can suppress the expression of proinflammatory inflammation factor, and suppress nuclear factor NF κ B receptor activations factor ligand (Receptor activator of nuclear factor kappa B ligand, RANKL) generation.Importantly, research observes that carnosic acid can suppress osteoclast generation and bone information in vitro, and treatment protection can be applied to joint injury in vivo, the effect of anti-sclerotin injury is played in the development of rheumatoid arthritis.Further biochemical analysis shows that carnosic acid can cause NFATcl downward with the RANKL Nuclear factor kappa B induced and MAPK (p38 and JNK) activation.The above results prove that carnosic acid may be developed to treat the medicine of patient with rheumatoid arthritis, with prevention of inflammation and joint injury.
Description
Technical field
The invention belongs to rheumatoid arthritis treatment technical field, more particularly to carnosic acid to close in treatment rheumatoid
Save the application in terms of scorching medicine.
Background technology
Rheumatoid arthritis is a kind of chronic auto-immune characterized by gradual synovial membrane inflammation and joint injury
Disease.Because rheumatoid arthritis people is to the complexity of therapeutic response, how to treat be still we face it is maximum
Problem.Non-steroid anti-inflammatory drug (NSAIDs) be clinically used to reduce inflammation, but its to prevent rheumatoid arthritis bone
The effect of damage, is weaker.Although alleviate modifying antirheumatic drug, including α-tumornecrosisfactor (TNF α) and interleukin-1 ' beta '
The inhibitor of (IL-1 β), it is a kind of effective and pretty good prospect treatment side to patient with rheumatoid arthritis to be proved to
Method.But serious infection can be caused or even can suffer from malignant tumour by taking in these medicines for a long time.Therefore, it is new to be badly in need of exploitation for we
The treatment rheumatoid arthritis supplement and alternative medicine of type, with prevention of inflammation and joint injury, reduce the production of adverse reaction
It is raw.
Rosemary (Rosmarinus officinalis L.) functions not only as flavouring, also serve as food, cosmetics,
The antioxidant of nutritious supplementary pharmaceutical and be widely used and be commercialized.Carnosic acid, it is that one kind is separated from rosemary leave
Major phenolic compound, it was found that in addition to its powerful antioxygenic property, carnosic acid also have important antibacterial,
The effect of anti-inflammatory, anticancer liver protection and chemoprophylaxis.Yu et al. (2009) researchs show that it is beta induced that carnosic acid can suppress IL-1
NF- κ B activation (Yu Y-M, Lin C-H, Chan H-C, Tsai H-D.2009.Carnosic acid reduces
cytokine-induced adhesion molecules expression and monocyte adhesion to
endothelial cells. European journal of nutrition 48(2)：101-106.).It is well known that NF-
κ B activation generates and pair particularly significant with disease caused by osteoclast excessive activation to osteoclast.Rheumatoid arthrosis
Caused by scorching bone erosion is osteoclast excessive activation, so inventor speculates, carnosic acid may to rheumatoid arthritis
In the presence of potential therapeutic action.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of carnosic acid to prepare treatment medicine for treating rheumatoid arthritis
The application of aspect.
In order to solve the above technical problems, the present invention uses following technical scheme：
Application of the carnosic acid in terms for the treatment of medicine for treating rheumatoid arthritis is prepared.
Application of the carnosic acid in terms for the treatment of Collagen-induced Arthritis medicine is prepared.
Application of the carnosic acid in terms of the anti-joint injury medicine for the treatment of rheumatoid arthritis is prepared.
Application of the carnosic acid in terms of the anti-inflammatory drug for the treatment of rheumatoid arthritis is prepared.
Have that expensive, toxic side effect is larger etc. for the medicine of existing treatment rheumatoid arthritis bone injury to ask
Topic, inventor combine the clinical practice of carnosic acid anti-inflammatory, and it may be invaded suppressing osteoclast generation and suppressing bone
Acted on caused by erosion, by inquiring into carnosic acid to the mouse osteoclast of in vitro culture, people's rheumatoid arthritis into fibre
The influence of the rat of cell sample synovial cell (RAFLS) and Collagen-induced Arthritis (CIA) is tieed up, it is found that carnosic acid is one
Kind is to treating the very promising native compound of rheumatoid arthritis.In vitro and in vivo studies have shown that, carnosic acid
The expression of proinflammatory inflammation factor can be suppressed, wherein proinflammatory inflammation factor includes α-tumornecrosisfactor (TNF α), interleukin 1
β (IL-1 β), interleukin-6 (IL-6), interleukin 8 (IL-8), Interleukin-17 (IL-17) and matrix
Metalloproteinases -3 (MMP-3), and suppress RANKL generation.Importantly, research observes carnosic acid in vitro
Osteoclast generation and bone information can be suppressed, and treatment protection can be applied to joint injury in vivo, in rheumatoid
The effect of anti-sclerotin injury is played in arthritic development.Further biochemical analysis shows that carnosic acid can also suppress
The nuclear Factor-Kappa B of RANKL inductions and the activation of MAPK (p38 and JNK), cause under NFATc1
Adjust.The above results prove that carnosic acid may be developed to treat the medicine of patient with rheumatoid arthritis, with prevention
Inflammation and joint injury.
Brief description of the drawings
Fig. 1 is that carnosic acid suppresses the broken of RANKL in bone induction in a manner of dose dependent
Bone cell differentiation and no cytotoxicity result of study figure.In figure：
(A) molecular structure of carnosic acid.
(B) carnosic acid can substantially suppress the differentiation of osteoclast.Handled with the carnosic acid of various concentrations, then use core
Factor-kappa B receptor activation factor ligand (100 nanograms/milliliter) stimulates the Osteoclast culture after 5 days (scale=100 micron)
The representative diagram of thing.
(C) quantitative analysis of osteoclast.To the Tartrate resistant acid phosphatase positive multinucleated cells (>=3 in (B) figure
Individual nucleus) counted.RANKL in bone processing, carnosic acid untreated fish group is compare, n=
3；*, P ＜ 0.05, * * * P ＜ 0.001.
(D) influence of the carnosic acid to derived from bone marrow macrophage vigor is detected by MTS.
(E) influence of the carnosic acid to RAW264.7 Apoptosis.RAW264.7 is incubated with the carnosic acid of various concentrations
Cell 24 hours, then carries out Apoptosis assay with flow cytometry.
Fig. 2 is that carnosic acid suppresses osteoclast function result of study figure.In figure：
(A) carnosic acid substantially suppresses the bone information ability of osteoclast.In the anti-tartaric acid acid of hydroxyapatite coating layer
Acid phosphatase dyes and the presentation graphics (engineer's scale=500 micron) of osteoclastic absorption.
(B) the quantitative analysis figure of Tartrate resistant acid phosphatase ion apocyte (>=3 nucleus) quantity.
(C) chart that the area absorbed using image J softwares measurement hydroxyapatite surface is formed, carnosic acid are untreated
Group is compares, n=3, * P ＜ 0.05, * * P ＜ 0.01.
(D) in real-time fluorescence quantitative PCR quantitative analysis osteoclast atomization marker genes expression.It is dense with difference
The carnosic acid processing of degree and 100 nanograms/milliliter RANKL in bone are incubated derived from bone marrow macrophage,
Detected after 5 days osteoclast specific gene courier ribonic acid (CTR (CTR), cathepsin K (CTSK) and
TRAP relative expression).Beta-actin is reference gene.Carnosic acid is untreated and the nuclear Factor-Kappa B receptor activation factor is matched somebody with somebody
Body treatment group is compares, n=3, * P ＜ 0.05, * * * P ＜ 0.001.
Fig. 3 is the mechanism analysis that carnosic acid suppresses osteoclast differentiation.In figure：
(A) Western blot detect influence of the carnosic acid to MAPK and NF- κ B signal paths.With carnosic acid (5
Micromole) pretreatment derived from bone marrow macrophage is after 1 hour, and then with RANKL in bone, (100 receive
Grams per milliliter) handle 0 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes and 60 minutes, crack protein simultaneously utilizes western blot
Method detects p-ERK1/2, total ERK1/2, p-p38, total p38, p-JNK, total JNK, I κ B α and beta-actin
Expression.
(B) quantitative analysis of protein expression.Relative expression is determined by photodensitometry.I κ B are determined using image J softwares
α bands are relative to the density of beta-actin band and each phosphorylation MAPK bands relative to its gross protein homologue
Ratio chart.N=3, * P ＜ 0.05, * * P ＜ 0.01, RANKL in bone processing and carnosic acid not
Treatment group is control.
(C) influence of the carnosic acid to NF- κ B transcriptional activities.NF- κ B fluoresceins are pre-processed with various concentrations carnosic acid
The RAW264.7 cells of enzyme reporter construct stable transfection 1 hour, then use RANKL in bone
Induction 6 hours.Measure NF- κ B uciferase activities.RANKL in bone is handled and carnosic acid is not located
The cell of reason is compares, n=3, * * P ＜ 0.01, * * * P ＜ 0.001.
Fig. 4 is the activation and calcium for the NFATc1 that carnosic acid suppresses RANKL in bone induction
Circumstances of oscillating study result figure.In figure：
(A) influence of the carnosic acid to NFAT transcriptional activities.Various concentrations carnosic acid handles NFAT luciferase reporters
The RAW264.7 cells of gene construct stable transfection 1 hour, then induce 24 with RANKL in bone
Hour.Measure NFAT uciferase activities.RANKL in bone is handled and carnosic acid is untreated thin
Born of the same parents is compare, n=3, * * P ＜ 0.01.
(B) Western blotting detection NFATc1 and c-fos expression.With carnosic acid (5 micromole) pre-process marrow come
Source macrophage 1 hour, RANKL in bone (100 nanograms/milliliter) is then used within the specified time
Stimulated.Extraction albumen simultaneously carries out the table of NFATc1, c-fos and beta-actin expression analysis, wherein beta-actin
Up to for internal reference.
(C) NFATc1 and c-fos quantitative analysis.Handled relative to RANKL in bone, mouse
The untreated control group of tail oxalic acid, determine that NFATc1 and c-fos bands move relative to β-flesh using Image J analysis softwares
The ratio of protein band.N=3, * P ＜ 0.05.
(D) influence that carnosic acid vibrates to RAW264.7 cell calciums.Experiment is divided into 3 groups：Only recombinate macrophage collection
G-CSF stimulate for negative control group (NEG)；Only RANKL in bone stimulate for the positive
Control group (POS)；RANKL in bone and carnosic acid (5 micromole) are jointly processed by group.Left figure is three
The representative picture of group Calcium Oscillations.Right figure is the quantitative analysis of calcium oscillation (maximum peak intensity subtracts baseline intensity, n=20).
Fig. 5 is rheumatoid arthritis-fibroblast sample synovial membrane that carnosic acid suppresses α-tumornecrosisfactor induction
The result of study figure of proinflammatory factor caused by cell.In figure：
(A) the inflammatory factor expression that Real-time PCR Analysis carnosic acid is induced α-tumornecrosisfactor.It is different
Carnosic acid processing rheumatoid arthritis-fibroblast sample synovial cell 1 hour of concentration, at α-tumornecrosisfactor
Reason is after 24 hours, real-time pcr fluorescence quantitative analysis a- TNFs (TNFa), interleukin-1 ' beta ' (IL-1 β), white thin
Born of the same parents' interleukin -6 (IL-6), interleukin 8 (IL-8), Interleukin-17 (IL-17), Transin-1 (MMP-
3) and RANKL in bone expression.Beta-actin encoding gene is as internal reference, α-tumor necrosis factor
Subprocessing and carnosic acid untreated fish group for control.N=3, * * P ＜ 0.01, * * * P ＜ 0.001.
(B) MTS detects influence of the carnosic acid to rheumatoid arthritis-fibroblast sample synovial cell's viability.
Fig. 6 is the development that carnosic acid significantly suppresses Collagen-induced Arthritis.In figure：
(A) rear solid end of C-II induced arthritis in rats representative photo of the 14th day after paresthesia epilepsy.
(B) influence of the carnosic acid to collagen-induced rat arthritis index score.The influence of sufficient pawl swelling；
(C) influence (D) carnosic acid of the carnosic acid to collagen-induced rat foot claw swelling is to collagen-induced rat body weight
Influence.
(E) generation of α-tumornecrosisfactor and interleukin-1 ' beta ' in different groups of ankle-joints is detected by ELISA.It is (strong
Health rat, n=6；Simple collagen induction group and medication therapy groups, n=8)
(F) by Western Blot analysis, the protein of RANKL in bone in the homogenate of measure joint
Horizontal (representative picture)；
(G) expression of RANKL in bone is quantitative (n=4).##P ＜ 0.01 and ###P ＜ 0.001,
Compared with healthy control group；* P ＜ 0.05 and * * P ＜ 0.01, compared with simple CIA model groups.
Fig. 7 is the destruction of joint result figure that carnosic acid significantly inhibits C-II induced arthritis in rats.In figure：
(A) X-ray shows influence (representative picture) of the carnosic acid to rat hind paw osteoclasia.
(B) to the iconography appraisal result of X-ray.
(C) ratio (BS/ by microcomputer sectional analysis joint bone volume (BV) between bone surface and bone volume
BV)。
(D) the representational 3-d reproduction of microcomputer tomoscan rear solid end is passed through.
(E) representative longitudinal stage casing of ankle-joint.
(F) the hematoxylin-eosin representativeness coloration result from different groups of rat ankle.
(G) to the appraisal result of pathology section.Methods of marking is shown in " materials and methods " part.##P ＜ 0.01 and ### P
＜ 0.001, compared with healthy control group；* P ＜ 0.05 and * * P ＜ 0.01, compared with simple CIA model groups.Every group of n=6-
8。
Embodiment
Osteoclast and fibroblast sample synovial cell and rats in vitro are collagen-induced in vivo for carnosic acid suppression
The arthritic inflammatory reaction of property and the research of destruction of joint
First, materials and methods
1. material and its source
Carnosic acid (purity >=98%) is bought in Man Site bio tech ltd (China, Sichuan Chengdu), is dissolved in
Dimethyl sulfoxide (DMSO).α-minimum must culture medium and hyclone buy in TRACE (Australia, Sydney).It is thin to recombinate macrophage
Born of the same parents' colony stimulating factor (M-CSF) and RANKL in bone albumen (RANKL) are bought in peace enlightening biology section
Skill Co., Ltd (U.S., Minneapolis).MTS reagent and luciferase assay reagent are from Pu Luomaige companies (the big profit of Australia
Asia, Sydney) in obtain.Antibody I κ B α, ERK, JNK, p38, phosphorylated (p) ERK, p-p38, p-JNK, and β-
Actin is bought in Santa Cruz Biotechnology (U.S., California, Dallas).Anti-NFATc1 and
Anti-c-fos antibody is bought in BD bioscience (U.S., California, San Jose).Antinuclear factor-κ B receptor activations
Factor ligand antibody is bought from prompt Science and Technology Ltd. (Britain, Cambridge) of liking to be beautiful.Tartaric-resistant kit
From Sigma-Aldrich trade Co., Ltd (Australia, Sydney) purchase.α-tumornecrosisfactor and interleukin 1
β ELISA reagents are bought in magnificent bioengineering Co., Ltd (China, Wuhan).Lyophilized chicken II Collagen Type VIs (CII) are bought in west
Ge Ma chemical companies (U.S., the Missouri State, St. Louis), and it is 3 mg/mls to be dissolved in concentration, temperature is the 0.05 of 4 DEG C
In mol/L acetic acid.
2. research and analyse method
2.1 external osteoclast generation analytic approach
The purpose of external osteoclast generation analysis is carried out, is to detect the shadow that carnosic acid breaks up to osteoclast
Ring.According to separate before us derived from bone marrow macrophage (BMM cells) method (Liu M, Xu L, Ma X, Xu J,
Wang J, Xian M, et al. (2015) .MAGED1 be mouse bone remodeling down regulator.《American Journal of Pathology》185
(10)：2653-2667), cell is positioned over concentration (to repeat three holes) in every 6000 BMMs/ orifice plates, cultivated 24 hours
Afterwards, carnosic acids of the BMM cells respectively with various concentrations is incubated, contains 100 nanograms/milliliter nuclear Factor-Kappas in culture medium
B receptor activations factor ligand and 30 nanograms/milliliters restructuring macrophage colony stimulatory factor (M-CSF).Nutrient solution is every other day
Change once.After five days, dyed using Tartrate resistant acid phosphatase, and calculate the Tartrate resistant acid phosphatase positive
Cell number (nuclear volume more than three is osteoclast).
2.2 cell activation assay methods
Carnosic acid is detected to BMM cells and rheumatoid arthritis-fibroblast sample synovial membrane using MTS analytic approach
The influence of cytoactive.For BMM cytoactives, according to 6*103Individual cells/well plate by the orifice plate of cell seeding tooth 96,
And carry out processing 48 hours with the carnosic acid (0,0.5,1,2.5,5,10,20 micromole) of various concentrations.Rheumatoid is closed
For the vigor for saving inflammation-fibroblast sample synovial cell, according to 5*103Individual cells/well by cell seeding in 96 orifice plates,
And it is incubated 48 hours with various concentrations carnosic acid (0,0.5,1,2.5,5,10,20 micromole).MTS is detected according to kit
Specification is carried out.With ELIASA (Bai Qi Science and Technology Ltd.s, Olten shellfish lattice, Germany) absorbance is determined in 490 nanometers.
2.3 use Apoptosis by Flow Cytometry
According to Annexin V-FITC apoptosis kit specification, (e-Bioscience, California, the Holy Land are sub-
Brother), handle RAW264.7 cells 24 hours with various concentrations carnosic acid (0,1,2.5,5,10,20 micromole), and use film
Even albumen-V and ofpropidium iodide solution dyeing.Flow cytometry analysis is carried out using CellQuest program analysis softwares,
Apoptotic cell in every group is expressed with percentage.
2.4 hydroxyapatite absorption processes
BMM cells are seeded in 6 orifice plates for being coated with collagen (density 1*105Individual cells/well) cultivated,
RANKL in bone (100 nanograms/milliliter) and restructuring macrophage colony stimulatory factor (30 nanograms/milli
Rise) stimulate 2 to 3 days.When osteoclast initially forms, equal number of osteoclast is simultaneously inoculated into by dissociated cell
On hydroxyapatite coating layer plate (CLS3989, the U.S. are healthy and free from worry).In restructuring macrophage colony stimulatory factor and nuclear Factor-Kappa B
In the presence of receptor activation factor ligand, cell is handled 48 hours with carnosic acid.Then, the hole of half is with 2.5% penta
Dialdehyde is fixed, and carries out tartaric-resistant, and remaining hole is bleached with 10% liquid lime chloride and removes cell.Light
Learn micro- sem observation, and the scope absorbed using Image J software analysis hydroxyapatite.
2.5 real-time fluorescence quantitative PCRs are analyzed
Using table of the real-time fluorescence quantitative PCR analytic approach detection osteoclast specific gene in the osteoclast of maturation
Up to level, rheumatoid arthritis-fibroblast sample synovial cell that cell factor induces in α-tumornecrosisfactor is detected
In expression.In order to detect the expression of osteoclast specific gene, by BMM cells according to 1*105Cells/well connect
Kind is in 6 holes, in the restructuring macrophage colony stimulatory factor and 100ng/ml nuclear Factor-Kappa B receptor activations of 30 nanograms/milliliters
In the presence of factor ligand, persistently handled 5 days with the carnosic acid (0,1,2.5,5 micromole) of various concentrations.WithKit (Life Science, Australia, Mulgrave) total ribonucleic acid (RNA) of extraction, and to ribose core
Sour (RNA) carries out complementary DNA synthesis and real-time fluorescence quantitative PCR analysis.The gene of quantitative analysis includes cathepsin K
(ctsk), CTR (CTR) and Tartrate resistant acid phosphatase (TRAP), carried out using beta-actin as internal reference relative
Expression analysis.The primer pair of said gene see document (Liu M, Xu L, Ma X, Xu J, Wang J, Xian M, et al.
(2015) .MAGED1 is the down regulator of mouse bone remodeling.《American Journal of Pathology》185(10)： 2653-2667).
In addition, we also have detected the expression of the inflammatory factor of α-tumornecrosisfactor stimulation.Rheumatoid is closed
Section inflammation-fibroblast sample synovial cell is planted in 12 orifice plates, with the carnosic acid (0,5,10 micromole) of various concentrations
After pretreatment 1 hour, α-tumornecrosisfactor (50 nanograms/milliliter) incubates 24 hours, extracts total ribonucleic acid (RNA) simultaneously
Carry out real-time fluorescence quantitative PCR analysis.Detection gene includes interleukin-6, interleukin 8, Interleukin-17,
Interleukin-1 ' beta ', α-tumornecrosisfactor, Transin-1 and RANKL, it is expressed as with beta-actin interior
Ginseng, detect the relative expression of above-mentioned each gene.Three parallel reactions of each experiment, in triplicate.
2.6 nuclear Factor-Kappa Bs (NF- κ B) and NFAT luciferase assays
The stable cell line RAW264.7 kinds that transfection has NF- κ B and NFATc1 are planted in 48 orifice plates, with various concentrations
Carnosic acid (0,1,2.5,5,10 micromole) pretreatment cell is handled 6 hours and 24 hours with RANKL respectively after 1 hour,
Then the detection of reporter gene is carried out using luciferase kit.
2.7 western blot analysis
Collect and according to 5*105The density of individual cells/well is inoculated with BMM cells in 6 orifice plates, respectively with containing carnosic acid and
After PBS without carnosic acid is handled 1 hour, RANKL (100 nanograms/milliliter) stimulates the different periods, is buffered in cracking
Cell is dissolved in liquid, wherein cracking, which is alleviated in liquid, contains 50mM Trise hydrochloric acid, 150mM sodium chloride, 5mMEDTA, 1%
Triton X-100,1mM sodium fluorides, 1mM sodium vanadate, 1% dexycholate, and protease inhibitor cocktail.Centrifugation
The protein in supernatant is collected, and carries out sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then will
Protein delivery is to nitrocellulose filter.Using immunoblotting assay method detect phosphorylated p38, phosphorylated CREB, phosphorylation JNK,
Total p38, total ERK1/2, total JNK, the level of I κ B, NFATc1, c-Fos and beta-actin.Finally, entered using image J softwares
Row gray analysis.
2.8 intracellular calcium oscillation measurements
Indicate to illustrate according to the method for manufacturer, studying calcium ion oscillations using calcium combination dye Fluo4-AM, (molecule is visited
Pin, the big profit of Australia, this sub- Coase, Thermo Fischer Scient Inc.).BMM cells are handled with carnosic acid (5 micromole) 1 hour,
Stimulated 24 hours using 100 nanograms/milliliter RANKL in the presence of macrophage colony stimulatory factor is recombinated afterwards.It is cloudy
Property and positive control are respectively simple M-CSF stimulations group and simple RANKL stimulations group.It is micro- using 5 when calcium ion oscillations measure
Mole calcium ion fluorescent (molecular probe, Australia, Victoria) and 0.05% pluronic F-127 calcium fluorescence
Probe (the silent winged generation that of match), temperature places cell 30 minutes indoors.It is warm indoors after determining buffer solution washing cell three times
Degree continues incubated cell 20 minutes, and is seen using the inverted fluorescence microscope (Nikon Ti-U) that excitation wavelength is 488 nanometers
Examine.Every 3 minutes capture images 2 seconds.Use the Nikon Basic Research software analysis of Nikon, research at least 2
The average peak height of the cell of individual vibration (peak-peak intensity subtracts baseline intensity).Calculate the thin more than 20 of each hole
Born of the same parents, calculate repeat three holes each time.
The structure of 2.9 Collagen-induced Arthritis models and the drug therapy of carnosic acid
Bought from 26 body weight are shared in 160 grams -180 grams female Vist mouse in the limited public affairs of Shanghai Si Laike experimental animals
Department (China, Shanghai) purchase female Wistar rats 26 (body weight is at 160 grams -180 grams or so).By rat feeding specific
Pathogen-free domestic (SPF) under the conditions of (22 DEG C, 12h/h light darks, 50%-55% humidity) and provide foods and water to them.
After the laundering period of one week, using with it is the 100 of Freund's complete adjuvant equal volume (U.S., Mu Cun, St. Louis, Sigma) micro-
Gram chicken II Collagen Type VIs (CII, the U.S., Mu Cun, St. Louis, Sigma) carry out intradermal injection immunity, now labeled as the 0th day.
After 7 days, the CII emulsified in incomplete Freund's adjuvant to rat skin lower injection equivalent.It is big to control group in the same way
Mouse injecting normal saline (n=6).When arthritic symptom breaks out (clinical arthritis index score >=2), arthritis will be suffered from
Rat be randomly divided into two groups, be model group and carnosic acid treatment group respectively.Carnosic acid treatment group is every other day in abdominal cavity
Interior injection carnosic acid (5mg/kg carnosic acid is dissolved in 0.9% physiological saline), model group every other day injects 0.9% life
Salt solution is managed, experimental period is 14 days.Starting for the 9th day after first time is immune, is commented using 0~4 clinical arthritis daily
Subsystem evaluates the clinical arthritic score of each hind leg：0=does not have arthritis；(IP) joint between one or two phalanges of 1=
Swelling is rubescent；(IP) joint suffers from arthritic or has a larger joint with pass between tri- or four phalanges of 2=
Section is scorching；3=, which exceedes, four joint redness or swelling；The whole claws of 4=suffer from serious arthritis, and every animal score is 0
Divide between -16.In addition, the oedema journey of claw is determined using sufficient pawl measuring instrument (YLS-7A, Shandong Academy of Medical Sciences equipment station)
Degree.Scoring and the measurement of sufficient pawl are implemented by two to being grouped unwitting tester.In addition, every is calculated after arthritis breaking-out
The change of disease rat body weight (%), formula are as follows：Changes of weight=[(the body of ill 15th day body weight/illness the 1st day
Weight) -1] * 100%.All zooperies are by the approval of the experiment committee of Nanjing Normal University.
In order to determine the cytokine levels of C-II induced arthritis in rats, ankle-joint is separated, by containing protease
Lysis buffer (the T-PER animal tissue protein extracts reagents of/inhibitor mixture；Sai Mo flies generation that) extraction tissue homogenate
Liquid, determine α-tumornecrosisfactor (TNF-α) with ELISA detection kit and interleukin-1 ' beta ' (IL-1 β) is horizontal.Such as
It is previously described, using Western Blot analysis, the RANKL expression in detection ankle-joint homogenate.
2.10 imaging evaluation
Using the Multifunctional small animal living imaging system of Kodak, CIA rat hindlimbs are observed by x-ray
Sufficient pawl.Parameter setting shoots for 35 kilovolts, 30 seconds time for exposure.According to document (Cai X, Zhou H, Wong YF, Xie Y,
Liu ZQ, Jiang ZH, et al. (2007) suppress the generation of Collagen-Induced Arthritis in rat by QFGJS and entered
Exhibition, the QFGJS is to come from Antiarthritic Chinese herbal medicine formula.《Medicine of national minorities magazine》10(1)：39-48) the scoring of report
System, evaluate destruction of joint degree.According to the scoring summation of the two of every rat rear solid ends, total iconography scoring is calculated, most
Big value is 6 points.Observer is unclear to packet situation.
2.11 histopathological scores
Sufficient pawl is fixed in 4% paraformaldehyde, the decalcification in 12% disodium ethylene diamine tetraacetate, and is embedded in paraffin
In.To section (4 microns) dye simultaneously om observation with hematoxylin-eosin (H＆E).Bibliography (Sims NA, Green
JR, Glatt M, Schlict S, Martin TJ, Gillespie MT, Romas are E.2004.Targeting
osteoclasts with zoledronic acid prevents bone destruction in collagen-
induced arthritis.Arthritis& Rheumatism 50(7)：2338-2346.), to bone erosion and Evaluation of Inflammation
Carry out arthritic histopathological evaluation.Scoring person is unclear to experiment packet situation.
2.12 microscopic CT scannings analysis bone is osteoclastic
Corroded to further analyze the osteopathy stove of ankle-joint, pass through microscopic CT scanning rat ankle joint (Micro-CT scanning, Bu Lu
Gram, Belgium, the hole base of a fruit is conspicuous) and carry out three-dimensional reconstruction.Our manual drawings close including gambrel and with the whole ankle including joint
Save as interest region (ROI), calculate the bone volume (BV) and bone surface (BS) in whole joint.In order to assess the part of bone surface
Corrode, calculating ratio BS/BV.
2.13 statistical analysis
As a result it is expressed as average ± the SD from the result for testing to obtain three times or more.Examined using the double tail t of non-matching
Carry out data statistic analysis.P ＜ 0.05 are considered to have statistical significance.
3rd, result
3.1 carnosic acids suppress differentiation and its function of the osteoclast of RANKL inductions in vitro
In order to study the influence that carnosic acid breaks up to osteoclast, by the BMM cells of culture and the rat-tail of various concentrations
Oxalic acid is incubated together, and TRAP dyeing is carried out after M-CSF and RANKL is stimulated 5 days, finds carnosic acid with dose dependent side
Formula substantially suppresses the differentiation (Figure 1B, C) of osteoclast.After the processing of 5 micromole's carnosic acids, only 50% osteoclast
Number form is into (Fig. 1 C).In order to exclude toxic effect of the carnosic acid to BMM cells, We conducted cell viability detection.Such as figure
Shown in 1D, carnosic acid will not also suppress the activity or propagation of BMM cells under up to 20 micromolar concentration.In the past
Research show that carnosic acid can induce the apoptosis of a variety of cancer cells, therefore, whether inventor investigates it to osteoclast point
Change has an impact.After RAW264.7 cells are handled 24 hours with a series of carnosic acid of concentration, and pass through fluidic cell instrument
Assess Apoptosis situation.As referring to figure 1E, carnosic acid nor affects on Apoptosis even in 20 micromoles, and this shows mouse
Tail oxalic acid is not as caused by Apoptosis to the inhibitory action of osteoclast.
After it specify that carnosic acid can suppress the differentiation of osteoclast, inventor is determined with hydroxyapatite absorption process
Whether carnosic acid can damage the function of osteoclastic bone resorption.As shown in figures 2 a-c, carnosic acid can be with dose-dependant
Mode significantly decrease hydroxyapatite re-absorption area, be reduced to 55% in 5 micromole.In order to further assess
Carnosic acid then have detected RANKL to osteoclast formation and the inhibitory action of function, inventor by real-time quantitative PCR
Osteoclast the marker gene CTR, CTSK and TRAP of induction mRNA expressions, as a result find, carnosic acid is significantly
Suppress a series of marker gene expression (Fig. 2 D) of osteoclast, this further demonstrates that carnosic acid is to osteoclast formation
Inhibitory action.To sum up result of study shows, carnosic acid suppresses the bone information function of the osteoclast of in vitro culture.
3.2 carnosic acids suppress MAPK the and NF- κ B of RANKL inductions activation
Further to explore the potential mechanism that carnosic acid suppresses osteoclast differentiation and activity, inventor passes through Diagnosis of Sghistosomiasis
Mark method have detected two important signal path MAPK and NF- κ B during osteoclast formation.As expected, RANKL stimulates 10
Minute, three MAPK family members, extracellular signal-regulated kinase (ERK), c-Jun N terminal kinases (JNK) and p38 swash
The phosphorylation level of enzyme substantially increases, and may persist to 30 minutes (Fig. 3 A, B).And carnosic acid can substantially suppress JNK's and p38
Phosphorylation (Fig. 3 A, B).Carnosic acid has not significant impact to the RANKL ERK induced phosphorylation level.
In addition to MAPK signal paths, inventor is also analyzed by the measure of Western blotting and luciferase reporter gene
Influence of the carnosic acid to NF- κ B signal paths.As shown in figs.3 a and 3b, after immunoblotting assay shows that RANKL is stimulated,
NF- κ B suppression subunit IkBa degraded rapidly in 10 minutes, but this degraded can substantially be pressed down by 5 micromolar carnosic acids
System.Inventor further utilizes NF- κ B luciferase reporter gene, it is found that carnosic acid can be in the form of dose-dependent
Suppress NF- κ B transcriptional activity (Fig. 3 C).
3.3 carnosic acids inhibit the NFATc1 activity that RANKL is induced
NFATc1 is differentiation and the major regulator of function of osteoclast.Inventor then passes through luciferase reporting
The influence for having tested and analyzed the NFATc1 activity that carnosic acid is induced RANKL of gene.As a result show, when RANKL is stimulated
During RAW264.7 cells, NFATc1 transcriptional activity increase.Carnosic acid significantly inhibits its activity in a manner of concentration dependent
(Fig. 4 A).In order to further inquire into the influence that carnosic acid is expressed NFATc1, right NFATc1 is analyzed using Western blotting
Protein level.As a result as shown in Figure 4 B and 4C, RANKL can significantly improve NFATc1 protein expression, and carnosic acid energy
Enough weaken NFATc1 protein level.It is well known that transcription factor c-fos is adjusted by NFATc1 startup factors
NFATc1 expression.Inventor then also have detected the expression of c-fos genes, as a result as shown in Figure 4 B and 4C, Salvia japonica
The sour obvious protein expression level for suppressing c-fos genes, this also further demonstrate what carnosic acid was induced RANKL
NFATc1 has expressed inhibitory action.
Intracellular calcium vibration be RANKL induction osteoclast atomization in NFATc1 activation necessary to
(Negishi- Koga T, Takayanagi H.2009.Ca2+-NFATc1 signaling is an essential axis
of osteoclast differentiation.Immunological reviews 231(1)：241-256.).In view of mouse
Tail oxalic acid then have detected carnosic acid in the cytoplasm that is stimulated by RANKL to the inhibitory action of NFATc1 activity, inventor
The influence of calcium ion oscillations.As a result show, carnosic acid can significantly inhibit RANKL induction calcium ion oscillations intensity, this with
Luciferase reporter gene detects and western blot analysis result is consistent (Fig. 4 D).
The processing of 3.4 carnosic acids significantly suppresses the rheumatoid arthritis in α-tumornecrosisfactor induction into fiber finer
The generation of the cell factor of born of the same parents sample synovial cell
Carnosic acid have proved to be a kind of very effective anti-inflammatory drug (Rau O, Wurglics M, Paulke A,
Zitzkowski J, Meindl N, BockA, et al. (2006) .Carnosic acid and carnosol, phenolic
Diterpene compounds of the labiate herbs rosemary and sage, are activators of
the human peroxisome proliferator-activated receptor gamma.Planta medica 72
(10)：881-887；
Kuo C-F, Su J-D, Chiu C-H, Peng C-C, Chang C-H, Sung T-Y, et al. (2011)
.Anti-inflammatory effects of supercritical carbon dioxide extract and its
isolated carnosic acid from Rosmarinus officinalis leaves.Journal of
agricultural and food chemistry 59(8)：3674-3685.).In order to determine carnosic acid whether in class wind
Also function to the effect of anti-inflammatory in wet arthritis, inventor have detected the rheumatoid arthritis of α-tumornecrosisfactor induction-
Cytokine profiles in fibroblast sample synovial cell are horizontal.As shown in Figure 5A, carnosic acid is with the side of concentration dependent
Formula substantially reduces the α-tumornecrosisfactor (TNF α) of α-tumornecrosisfactor mediation, and interleukin-1 ' beta ' (IL-1 β) is white thin
Born of the same parents' interleukin -6 (IL-6), interleukin 8 (IL-8), Interleukin-17 (IL-17) and Transin-1
(MMP-3) expression.In addition, RANKL is osteoclast differentiation and activates most important regulatory factor, inventor, which detects, to be found,
Carnosic acid can also significantly inhibit RANKL expression.MTS detection cell viabilities find that carnosic acid is even in up to
During 20 micromole, (Fig. 5 B) is not influenceed on rheumatoid arthritis-fibroblast sample synovial cell survival rate.
Influence of 3.5 carnosic acids to arthritis severity and to Arthritis development process
It is that further to the possibility therapeutic effect of rheumatoid arthritis, inventor establishes collagen-induced clear and definite carnosic acid
Rheumatoid arthritis rat model.Those have been suffered from into arthritic rat (clinical arthritis index score >=2) to be divided into
Two groups, one group is every other day injected carnosic acid (5 mg/kgs, being dissolved in 5% DMSO/95% PBS), and another group only
The DMSO/95% of injection 5% PBS, 14 days are a phase.As shown in Figure 6A, 5% DMSO/95% PBS collagen is injected
Induced Arthritis rat can substantially observe the arthritic symptom of such as erythema and swelling, and carnosic acid can be notable
Weaken the arthritic order of severity.Compared to control group, carnosic acid drug-treated group rat arthritis index score is up to
4.2, reduce 30% (Fig. 6 B).Sufficient pawl measurement result has also further demonstrated that this point (Fig. 6 C).Due to body weight mitigation with
The clinic of rheumatoid arthritis is actively in progress relevant, and for this, inventor have detected the change of body weight after arthritic.Such as figure
6D, carnosic acid treatment group can effectively suppress the mitigation of C-II induced arthritis in rats body weight.Exist for further evaluation
Antiinflammatory action in C-II induced arthritis in rats, inventor have detected α-tumornecrosisfactor in rat ankle joint homogenate
The change of (TNF α) and interleukin-1 ' beta ' (IL-1 β), as a result finds：Carnosic acid can be significantly inhibited in joint tissue
TNF α and IL-1 β synthesis (Fig. 6 C).Western blot analysis result shows that carnosic acid also reduces RANKL eggs in joint tissue
The synthesis of white matter, this point are consistent with Vitro Experimental Results.The reduction of RANKL caused by carnosic acid, may be to collagen-induced
Property arthritic animals osteoclasia brings certain protective effect (Fig. 6 F, G).
The processing of 3.6 carnosic acids can suppress the osteoclasia of Collagen-induced Arthritis animal
Using X ray, Micro-CT scanning and histological score, it is big to Collagen-induced Arthritis that inventor have studied carnosic acid
The influence of mouse destruction of joint.Under the conditions of x-ray imaging, without arthritic healthy rat show normal articulation structure and
Joint space.Rats with arthritis without drug-treated shows have typical destruction of joint, Joint shift and joint space
Narrow rheumatoid arthritis (Fig. 7 A).However, radiology testing result and mean radiological fraction show, Salvia japonica
Destruction of joint in acid treatment group rat is significantly suppressed (Fig. 7 A, B).Micro-CT scanning is also further shown, with healthy control group
Rat is compared, and the ankle-joint surrounding bone display bone volume of C-II induced arthritis in rats substantially reduces (BV) and parameter amount
The superficial density increase (BS/BV) of change, and the rats with arthritis of carnosic acid processing can then suppress bone volume and bone surface with
The change of ratio between bone volume, show that carnosic acid has the potential protective effect (figure prevented in focal bone erosion
7C).In addition, histopathological scores also indicate that, suffering from has synovial hyperplasia, inflammatory cell in arthritic rat ankle joint
The pathological characteristicses such as infiltration, cartilage damage and bone erosion (Fig. 7 F), and the cunning of rat its ankle-joint after carnosic acid processing
Film inflammation and osteoclasia show significantly to improve (Fig. 7 F, G).
4th, discuss
Carnosic acid, it is the content highest antioxidant found in the leaf of rosemary, except it is proven to have
Outside antioxidation activity, it also plays strong anti-inflammatory property in many cells and animal model.RAU et al. (2006)
The antiphlogistic effects of carnosic acid are attributed to its activation (Kuo C- to peroxisome proliferators activated receptor γ
F, Su J-D, Chiu C-H, Peng C-C, Chang C-H, Sung T-Y, et al. (2011) .Anti-inflammatory
effects of supercritical carbon dioxide extract and its isolated carnosic
acid from Rosmarinus officinalis leaves.Journal of agricultural and food
chemistry 59(8)：3674-3685；
Rau O, Wurglics M, Paulke A, Zitzkowski J, Meindl N, Bock A, et al. (2006)
.Carnosic acid and carnosol, phenolic diterpene compounds of the labiate herbs
Rosemary and sage, are activators of the human peroxisome proliferator-
activated receptor gamma.Planta medica 72(10)： 881-887).Separately there are some researchs to show, rat-tail
Oxalic acid can by suppressing cell factor and chemotactic factor (CF) such as α-tumornecrosisfactor, interleukin-1 ' beta ', interleukins-
6, interleukin 8, MCP-1, CCL5, CXCL10, play straight in the macrophage and keratinocyte that LPS is stimulated
Meet antiinflammatory action (Tsai T-H, Chuang L-T, Lien T-J, Liing Y-R, Chen W-Y, Tsai P-J (2013)
.Rosmarinus officinalis Extract Suppresses Propionibacterium acnes-Induced
Inflammatory Responses.Journal of medicinal food 16(4)：324-333.).Based on conventional sight
Examine, inventor infers, carnosic acid may weaken the order of severity of rheumatoid arthritis by anti-inflammatory.Inside and outside is real
Test and also demonstrate this point, material evidence is as follows：1) carnosic acid inhibits the arthritis synovial fibroblast that TNF α induces
The secretion of a variety of inflammatory factors, including α-tumornecrosisfactor, interleukin-1 ' beta ', interleukin-6, interleukins-
8th, Interleukin-17 and Transin-1 (MMP-3)；2) carnosic acid has lowered rats with arthritis ankle-joint
The synthesis of α-TNF and interleukin-1 ' beta ' in homogenate；3) carnosic acid reduce in arthritis synovial cell and
RANKL expression in collagen-induced rat；4) carnosic acid reduces collagen-induced property in clinical and histopathology level
The inflammatory reaction of rats with arthritis.In a word, antiinflammatory action of the carnosic acid in rheumatoid arthritis, and in the past at other
The result of study of the carnosic acid of diseases associated with inflammation is similar.In other words, anti-inflammatory of the carnosic acid in rheumatoid arthritis is made
With the inhibitory action being embodied in a series of expression of inflammatory cytokines.
In rheumatoid arthritis treatment, one of most important clinical problem is gradual joint injury, this and work(
Energy sexual dysfunction is closely related.Although most of resisting rheumatoid arthritis medicines successfully improve arthritis, they
Prevent that there is the effect of less in terms of destruction of joint.In present study, inventor proves that carnosic acid can not only for the first time
Suppress arthritis, and external osteoclast differentiation and internal osteoclasia can also be suppressed.Osteoclast is responsible for bone information, is
The main effects cell of joint injury.In vitro study shows that carnosic acid can significantly inhibit the osteoclast of RANKL inductions
Differentiation and bone resorption activity, and this inhibitory action is bred with cell and apoptosis does not have any relation.With results of in vitro studies
Consistent, intra-body data shows, iconography, which scores, microscopic CT scanning is consistent with histopathological scores shows, carnosic acid shows
Work reduces the joint injury as caused by Collagen-induced Arthritis.Inventor further speculates, right possessed by carnosic acid
External osteoclast generation and the inhibitory action of internal osteoclasia, it may be realized by suppressing NF- κ B and MAPK activity.
It is well known that the NF- κ B paths of RANKL mediations are particularly significant in the formation and activation of osteoclast.RANK and
RANKL interphase interaction, cause the release of fast degradation and subsequent NF- κ B that I κ B pass through proteasome, NF- κ B displacements
Into core and start the transcription of osteoclast marker gene.In our current research, inventor has found, carnosic acid substantially suppresses
NF- κ B signal paths, show as I κ B α degraded and NF- κ B transcriptional activation level is suppressed.Except NF- κ B believe
Number path, three MAPK family members ERK, JNK and p38 of RANKL inductions activation break up activation and existence in osteoclast
In also play an important role.The study find that carnosic acid can suppress the JNK and p38 of RANKL inductions phosphorylation
It is horizontal.However, ERK phosphorylation is not suppressed by carnosic acid.These as shown by data, carnosic acid may pass through a plurality of letter
Number path suppresses the differentiation of osteoclast, and its direct action target spot still needs to further disclose.
It is well known that NFATc1 is located at the NF- κ B and MAPK signal paths downstream of RANKL inductions, it is osteoclast differentiation
With the transcription factor of most critical in activation.In present study, the NFATc1 of RANKL inductions protein expression and transcription
Activity is by carnosic acid significantly inhibits.And carnosic acid also substantially inhibits NFATc1 upstream gene c-fos's
Protein expression, downstream target gene TRAP, CTR and CTSK be also significantly down-regulated, numerous evidences unanimously demonstrate NFATc1 in mouse
Tail oxalic acid suppresses to play key player in osteoclastic atomization.
Document shows, NFATc1 can also adjust calcium ion in osteoclastogenesis release (Negishi-Koga T,
Takayanagi H(2009).Ca2+-NFATc1 signaling is an essential axis of osteoclast
differentiation. Immunological reviews 231(1)：241-256.).This research research discovery, Salvia japonica
The intracellular calcium that acid significantly reduces RANKL inductions is horizontal, and this also demonstrate again NFATc1 in the process important
Effect.To sum up, carnosic acid is discharged by NF- κ B, the MAPK and calcium ion for suppressing RANKL inductions, causes NFATc1 expression
And activity down-regulation, so as to cause the differentiation of osteoclast and activation to be suppressed., it is known that inflammation can cause osteoclast to generate,
And cause the generation of rheumatoid arthritis bone information.In this project, inventor has found that carnosic acid can effectively suppress
The generation of a series of proinflammatory inflammation factor, and lower RANKL expression.The reduction of these Cytokine Expression Levels, may
Suppress formation and the bone resorption activity of osteoclast.Importantly, cell and molecular data show, carnosic acid can lead to
Cross the activation directly differentiation of suppression osteoclast and its function for suppressing NF- κ B and MAPK.Therefore, the anti-bone of carnosic acid is broken
Bad effect may be attributed to indirectly-acting caused by the direct repression and suppression inflammation of osteoclast formation.
In addition to effect, the safety problem of medicine it is also contemplated that.It is well known that carnosic acid is peace for the health of the mankind
Complete.Food and drug administration and EFSA have been approved by, and carnosic acid can as antioxidant
It is widely used in including oil, animal fat, sauce, baking goodses, meat and fish products in a series of food of class.This
In project, 5 micromolar carnosic acids can suppress the mitigation of C-II induced arthritis in rats body weight；And up to 20
BMM cells and the cell viability of rheumatoid arthritis-fibroblast sample synovial cell are nor affected on during micromole, this says
The security of bright carnosic acid is high.In view of its validity and security, carnosic acid is a kind of up-and-coming treatment class wind
Wet arthritic supplement or alternative medicine.
Claims (4)
1. application of the carnosic acid in terms for the treatment of medicine for treating rheumatoid arthritis is prepared.
2. application of the carnosic acid in terms for the treatment of Collagen-induced Arthritis medicine is prepared.
3. application of the carnosic acid in terms of the anti-joint injury medicine for the treatment of rheumatoid arthritis is prepared.
4. application of the carnosic acid in terms of the anti-inflammatory drug for the treatment of rheumatoid arthritis is prepared.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115737617A (en) * | 2022-11-18 | 2023-03-07 | 中国人民解放军总医院第五医学中心 | Application of carnosic acid or derivatives thereof in preparing medicament for treating and/or preventing type I interferon |
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| CN115737617A (en) * | 2022-11-18 | 2023-03-07 | 中国人民解放军总医院第五医学中心 | Application of carnosic acid or derivatives thereof in preparing medicament for treating and/or preventing type I interferon |
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