CN109846885A - The new application of Rosuvastatin - Google Patents

Abstract

The present invention provides the new application of Rosuvastatin (Rosuvastatin), the Rosuvastatin is for promoting dental pulp stem cell (Dental Pulp Stem Cells, DPSCs) to break up at dentine.According to the present invention, Rosuvastatin is by inhibiting NF- κ B signal signal pathway activated, so that DPSCs be promoted to break up at dentine, can provide new scheme for treatment dental caries.

Description

The new application of Rosuvastatin

Technical field

The present invention relates to the new applications of Rosuvastatin (Rosuvastatin), and specifically Rosuvastatin is for promoting Break up into dental pulp stem cell (Dental Pulp Stem Cells, DPSCs) at dentine.

Background technique

Dental caries (Dental Caries) and tumour, hypertension are listed as cause to seriously endanger to human health three by WHO Big common disease, and dental caries are classified as to the third position non-communicable diseases of global range domestic demand keypoint control, untreated permanent teeth Dental caries are affected more than world's one third population.Largely studies have shown that dental caries were fallen ill as a kind of multifactorial disease Journey is caused by the dynamic equilibrium of pathology sexual factor and protectiveness factors composition is broken.The generation of dental caries, mostly anaerobic bacteria are Main gram-positive bacterium and gramnegative bacterium mixed infection, wherein streptococcus mutans (S.mutans) are current dental caries Generally acknowledged main pathogenic bacteria.Streptococcus mutans by mycoprotein and lecithin effect ultimately form complicated biofilm structure with Continually changing oral environment is adapted to, release lactic acid makes enamel decalcification and causes inflammatory cell infiltration and eventually lead to dentine Destruction.Although body can generate reparative dentin to maintain tooth body structure under conditions of bacterium and inflammatory factor stimulate Integrality, but due to the breakdown speed of dentine far beyond its repair speed it is fast, eventually lead to the generation of dental caries, or even cause Pulpitis.Therefore, the dentine repair mechanism under dental caries state is fully realized, the prevention and clinical treatment to the disease have far-reaching Meaning.

Rosuvastatin (Rosuvastatin) is a kind of selective 3-hydroxy-3-methylglutaric acid list acyl coenzyme (A3- Hydroxy-3-methylglutarylcoenzyme A, HMG-CoA) reductase inhibitor, wide clinical application is in reduction gallbladder Sterol treats and prevents cardiovascular disease.It is nearest the study found that Rosuvastatin body cardiovascular system, immune system, There is multiple-effect effect in major system such as skeletal system.It is interesting that more and more researches show that, statins can be effective Reduce tumor necrosis factor-alpha (Tumor Necrosis Factor α, TNF-α), interleukin-11 β in level in gingival sulcus fluid The expression of the inflammatory factors such as (Interleukin-1 β, IL-1 β), interleukin 6 (Interleukin 6, IL-6).Meanwhile Rosuvastatin, because its slow-release capability has significant anti-inflammatory effect, can efficiently inhibit c-Jun as statins of new generation Terminal Kinase (c-Jun Terminal Kinase, JNK), nuclear factor kappa B (Nuclear Factor Kappa B, NF- κ B activation and reduction interleukin 8 (Interleukin 8, IL-8), IL-6, MCP 1 (Monocyte) Chemoattractant Protein 1, MCP-1), Intercellular surface adhesion molecule-1 (Intercellular Adhesion Molecule 1, ICAM-1) and cyclooxygenase-2 gene (Cyclooxygenase 2, COX-2) expression.Based on previous scholars' Research achievement, the invention reside in the inflammatory reactions how discussion Rosuvastatin participates in external dental caries cell model.

It is well known that nuclear factor-kappa B (Nuclear Factor-Kappa B, NF- κ B) in inflammatory reaction and It is played a crucial role in terms of cells survival.NF- κ B is present in cytoplasm, be made of I κ B α, p50 and p65 it is different Tripolymer.When tripolymer activation, I κ B α is degraded, and release NF- κ B enters core in conjunction with special DNA adjusting sequence, thus Adjust the transcription of gene.The proinflammatory processes of dental caries are related with NF- κ B signal Pathway Activation, cause dental caries early stage to have abnormal Inflammatory factor release.The inflammatory factor release the study found that inhibition NF- kB activity can decay in recent years, and promote DPSCs at tooth Essence differentiation and Mineral nodules are formed.Therefore, the invention reside in research Rosuvastatin regulation DPSCs at dentine differentiation whether It is suppressed with NF- κ B signal access related.

Summary of the invention

Technical solution to be solved by this invention is to provide the new application of Rosuvastatin.

Specifically, the use the present invention provides Rosuvastatin in the disease for the treatment of dentine or dentin matrix On the way.

Further, disease includes saprodontia or hypoplasia.Hypoplasia can be hypoplasia of dentin.

Further, Rosuvastatin is by promoting DPSCs at dentine differentiation therapy disease.

Further, Rosuvastatin is in inflammatory microenvironment, by inhibiting NF- κ B signal access to promote DPSCs at tooth Essential differentiation therapy disease.

Further, Rosuvastatin first choice concentration is 1-30 μM.

Further, Rosuvastatin first choice concentration is 5-25 μM.

Further, Rosuvastatin first choice concentration is 8-20 μM.

Further, Rosuvastatin first choice concentration is 10 μM.

The present invention establishes external DPSCs into dentine differentiation model, and lipopolysaccharides is used alone (Lipopolysaccharide, LPS) or use stimulation DPSCs together with Rosuvastatin.Firstly, we are contaminated using alizarin red Color, ALP dyeing, RT-PCR, Western Blot technology, DPSCs is at dentine differentiation capability after analysis Rosuvastatin stimulation And Mineral nodules formational situation.Secondly, Western Blot technology detects Rosuvastatin to DPSCs inflammation in inflammatory microenvironment The inhibition level of the factor (TNF-α, IL-1 β, IL-6) release.Finally, Western Blot and immunofluorescence dyeing measure NF- κ B Activation.Our research indicate that Rosuvastatin promotes DPSCs to break up at dentine by inhibition NF- κ B signal access, New understanding is provided for the Odontogenic cysts differentiation of DPSCs.

Detailed description of the invention

Fig. 1 is the present invention experimental result that Rosuvastatin promotes DPSCs to break up at dentine in noninflammatory microenvironment Figure；

Fig. 2 is the present invention experimental result picture that Rosuvastatin promotes DPSCs to break up at dentine in inflammatory microenvironment；

Fig. 3 is the experimental result picture for the inflammatory factor release that Rosuvastatin of the present invention inhibits LPS induction；

Fig. 4 is that NF- κ B signal access of the present invention is closed in Rosuvastatin promotion DPSCs at performance in dentine atomization The experimental result picture of keyness effect.

Specific embodiment

Illustrate with reference to the accompanying drawing, invention is further described in detail.

Present invention research Rosuvastatin is to DPSCs at the influence of dentine differentiation capability.

Experimental method:

One, N-DPSCs and I-DPSCs separation and culture:

(1) DPSCs in experimentation matches from clinically normal and dental caries tooth, Gender, age, Foundation-free disease is obtained into group personnel's informed consent, and ratify through Hospital Attached to Nantong Univ.'s Medical Ethics Committee.

(2) the normal and dental caries bad tooth pulled out is immediately placed in DMEM culture solution (penicillin containing 100U/mL/100 of pre-cooling μ g/mL streptomysin).

(3) Iodophor teeth surfaces, the rongeur edge after being sterilized with high pressure steam sterilization are used on Medical purification workbench Enamelo-cemental junction opens pulp cavity, exposure pulp chamber.

(4) dental pulp to get off will be cut to be placed in 3mg/mL Type I collagen enzyme solutions, shreds tissue and is placed in 37 DEG C of baking ovens Digestion 1 hour obtains single cell suspension through 70 μm of cellular metal net filtration.

(5) single cell suspension is inoculated in 25cm2 culture dish, be added comprising 10%FBS, DMEM, 1% glutamic acid and 100U/mL penicillin/100 μ g/mL streptomysins complete medium.

(6) culture dish is put into 37 DEG C of 5%CO2 incubator cultures, changes liquid every three days.

(7) 7-10 days after being about inoculated with, cell fusion reaches 80%-85%, with the passage of 1:3 ratio；

(8) it is named as N-DPSCs from the DPSCs that healthy tooth separates, the DPSCs separated from deep dental caries tooth is named as I- DPSCs, the third generation to the 5th generation DPSCs are used for following all experiments.

Two, external DPSCs is established into dentine differentiation model:

(1) take P3 for DPSCs with 2 × 104/ware density be inoculated in into dentine differential medium (15%FBS, DMEM, 10mmol/L β-glycerophosphate, 0.292mg/mL glutamine, 50mg/mL α-ascorbic acid, 10nmol/L dexamethasone, 100U/mL penicillin/100 μ g/mL streptomysins) culture, it changes within every 2 days liquid 1 time, breaks up 21 days.

(2) cell is collected, detection breaks up marker protein (DMP1, DSPP) at dentine, specifies DPSCs and break up at dentine Model foundation success.

(3) with the Rosuvastatin of various dose (0,0.1,1.0,10 and 100 μM, AstraZeneca, Britain) and (or) LPS (20 μ g/mL, Sigma, the U.S.) and (or) BMS-345541 (Sigma, the U.S.) stimulation are collected after stimulation 6 hours and 21 days Cell；

Three, Alizarin red staining:

(1) 30 minutes are fixed with 4% paraformaldehyde after the DPSCs that PBS washing induction is broken up at dentine.

(2) PBS cleans remaining paraformaldehyde liquid, and alizarin red dye liquor (pH=4.2) is added and dyes 10 minutes.

(3) dye situation is observed after cleaning dye liquor and is photographed to record.

Four, ALP is dyed:

(1) 30 minutes are fixed with 4% paraformaldehyde after the DPSCs that PBS washing induction is broken up at dentine.

(2) PBS cleans remaining paraformaldehyde liquid, is added and dyes 30 points by the configured working solution of kit specification Clock.

(3) dye situation is observed after cleaning dye liquor and is photographed to record.

Five, Western Blot:

(1) total protein of cell extracts

Culture medium is exhausted, three times, cell scraper scrapes de- cell to the PBS washing cell of pre-cooling, collects to the centrifuge tube of pre-cooling, 4 1000rpm is centrifuged 5 minutes under the conditions of DEG C, and the cell after washing is transferred to the clean EP pipe of pre-cooling；Operation is prepared cell and is split on ice Liquid is solved, 10 μ L inhibitors of phosphatases, 1 μ L protease inhibitors, 5 μ L 100mM PMSF are added in 1mL Lysis Buffer, is mixed Several minutes are saved after even on ice for use；The ratio of 1mL cell pyrolysis liquid is added according to every 107 cells in the cell washed Cell pyrolysis liquid is added, 200 μ L pipettors are blown and beaten repeatedly, until albumen is precipitated；4 DEG C of shaking tables, mild shaking 15 minutes；4 DEG C of conditions Lower 12000rpm is centrifuged 5 minutes, abandons supernatant, and -80 DEG C of low temperature refrigerators save.

(2) caryoplasm separates: extracting cytoplasm protein and Nuclear extract respectively

Cell normally is collected, culture medium is carefully drawn, blots as far as possible；Cell is washed twice with pre-cooling PBS, is washed every time Blot supernatant as far as possible afterwards；The 200 μ L that pre-cooling is added in cell precipitation are added to the cytoplasm extracting solution A of PMSF, and oscillation is mixed Even or piping and druming mixes, and sets and vibrates 30 minutes on ice；Under the conditions of 4 DEG C, 12000rpm is centrifuged 5 minutes, by one pre-cooling of supernatant sucking Clean EP centrifuge tube in get endochylema component is arrived, -80 DEG C of low temperature refrigerators save；The PBS of precipitating pre-cooling washed once, so Afterwards under the conditions of 4 DEG C, 12000rpm is centrifuged 5 minutes, abandons supernatant；The Nuclear extract that 50 μ L of addition pre-cooling are added to PMSF mentions Liquid B is taken, oscillation mixes or piping and druming mixes, and sets and vibrates 30 minutes on ice；Under the conditions of 4 DEG C, 12000rpm is centrifuged 10 minutes, will be upper To get karyon component is arrived in the clean EP centrifuge tube of clear one pre-cooling of sucking, -80 DEG C of low temperature refrigerators are saved.

(3) gel electrophoresis and Western Immuno detection

The preceding cleaning of glass plate, preceding, slab guarantees that bottom flushes and clips glass plate rear thin plate；Ultrapure water is added Between glass plate, whether observation liquid level declines, if liquid level does not decline, water is poured out, is blotted residual water with filter paper；First with separation Glue finally plus after TEMED mixes rapidly, is slowly added to separation gel with the liquid-transfering gun of 1000 μ L, pays attention to cannot thering is bubble, in arrival Until frame baseline；Isopropanol sealing is used on glue, after waiting gelling in 30 minutes or so, isopropanol is poured out, is rushed with ultrapure water It is extremely tasteless, residual water is blotted with filter paper；Again with concentration glue, after remaining space is filled up, comb is plugged；Wait gelling Afterwards, glue is removed together with plate；Suitable electrophoresis frame and slot are selected, offset plate is installed closely, electrophoresis liquid is leaked hunting；Boiled egg is white, Survey protein concentration, sample-adding；Marker is from left to right successively loaded in left side；Electrophoresis liquid there was not bottom to form electric current in outer box Access；Suitable lid is selected, power supply, constant pressure 80V are connected；Every 10-15 minutes observation is primary, when finding that Marker runs away, It is changed to the continuation of 120V voltage；Glue is cut, glue, pvdf membrane, filter paper will keep wet, there cannot be bubble on glue, and pvdf membrane is first put into first Depolarized in alcohol, after be put into ultrapure water, finally in transferring film liquid by cotton pad, filter paper, adhesive tape, pvdf membrane, filter paper, cotton pad it is suitable Sequence is put；Entire transferring film box is put into ice water, transferring film condition is set as constant current 350mA, 90 minutes；By PVDF after transferring film Film is put into confining liquid, is put into shaking table at room temperature and is closed at least 2 hours；Confining liquid is discarded, TBST is washed film 5 minutes × 3 times；Phase Primary antibody is answered to be incubated for, 4 DEG C of refrigerators save overnight；TBST is washed film 10 minutes × 3 times；Secondary antibody is incubated for 2 hours, and TBST washes 15 minutes × 3 It is secondary；Film is developed.

Six, immunofluorescence dyeing:

(1) DPSCs being inoculated in 24 orifice plates added with sequin, 37 DEG C of overnight incubations, density is about 1 × 105/ Hole.

(2) original culture medium is discarded, cells rinsed with PBS 1 time, fixes 40 minutes with 4% paraformaldehyde room temperature, PBS Washing 10 minutes × 3 times.

(3) sequin is chosen and is placed in wet box, confining liquid (0.1%TritonX-100) in drop, room temperature is closed 1 hour.

(4) reject confining liquid is not washed, directly dropwise addition primary antibody (β-catenin, p65), 4 DEG C of overnight incubations, and rewarming 0.5 is small When, PBS is washed 5 minutes × 3 times.

(5) it is protected from light and fluorescence secondary antibody is added, 4 DEG C are incubated for 2 hours, and PBS is washed 5 minutes × 3 times after rewarming.

(6) DAPI contaminates core, and 4 DEG C are incubated for 2 hours, and PBS is washed 10 minutes × 3 times after rewarming.

(7) under the conditions of being protected from light, mounting liquid is added dropwise on glass slide, sequin is tipped upside down on confining liquid, is careful not to produce Anger bubble is finally placed in fluorescence microscopy under the microscope.

Experimental result

1, as shown in Figure 1, Rosuvastatin promotes DPSCs to break up at dentine in noninflammatory microenvironment:

To inquire into the influence that the Rosuvastatin of various concentration breaks up DPSCs, we are contaminated by Alizarin red staining and ALP Color discovery, 10 μM of Rosuvastatin group can obviously increase the formation of Mineral nodules.Therefore, we choose 10 μM and grind as subsequent The optium concentration (Fig. 1) studied carefully.

At dentine induction 21 days after (0,0.1,1.0,10 and 100 μM) stimulation DPSCs of Rosuvastatin.(A) alizarin Plain red colouring and ALP staining analysis Mineral nodules formational situation.

2, as shown in Fig. 2, Rosuvastatin promotes DPSCs to break up at dentine in inflammatory microenvironment:

We distinguish separating-purifying DPSCs, Alizarin red staining knot from the tooth and healthy tooth that clinical diagnosis is deep dental caries Fruit, which shows to be formed in I-DPSCs group Mineral nodules, to be significantly reduced.Similarly, ALP dyeing also reflects identical result (Fig. 2A).Table Bright I-DPSCs is impaired at dentine differentiation capability.Then, DPSCs is stimulated using LPS, simulates a kind of Streptococcus mutans (gram Positive bacteria is most important pathogen in saprodontia generating process) caused by depth dental caries microenvironment, observe Rosuvastatin to DPSCs The influence broken up at dentine.After cultivating 3 weeks, more Mineral nodules (figure is formed compared with the control group at dentine differentiation (OD) 2B).This shows into dentine induction condition and plays a crucial role at dentine atomization.At the same time, with OD group It compares, LPS can significantly reduce the formation of Mineral nodules, and Rosuvastatin can obviously reverse the inhibiting effect (Fig. 2 B) of LPS.

N-DPSCs, I-DPSCs are respectively at dentine induction 21 days.(A) Alizarin red staining and ALP staining analysis mine Change tubercle formational situation.Rosuvastatin (10 μM) and (or) LPS (20ng/mL) stimulation DPSCs after at dentine induction 21 days.(B) Alizarin red staining and ALP staining analysis Mineral nodules formational situation.

3, as shown in figure 3, Rosuvastatin inhibits the inflammatory factor release of LPS induction:

In order to measure the regulating power that Rosuvastatin discharges DPSCs inflammatory factor, Western Blot analysis is found, The TNF-α that Rosuvastatin can effectively inhibit LPS to induce, the protein expression (Fig. 3) of IL-1 β and IL-6.These results show that Rosuvastatin energy effective attenuation inflammatory reaction.

Rosuvastatin (10 μM) and (or) LPS (20ng/mL) are stimulated after DPSCs complete medium culture 6 hours.(A) After Western Blot analyzes LPS stimulation, proinflammatory cytokine (TNF-α, IL-1 β and IL-6) expression is obviously increased, and Rosuvastatin can effectively inhibit this phenomenon.

4, as shown in figure 4, NF- κ B signal access promotes DPSCs at playing in dentine atomization in Rosuvastatin Key effect:

NF-kB is an important transcription factor in inflammatory process, and existing whether we detect Rosuvastatin to this mistake Journey has inhibiting effect.Analysis finds that LPS can promote p65 phosphorylation, I κ B α degradation and p65 nuclear transfer, and Rosuvastatin can press down Make this process (Fig. 4).Immunofluorescence dyeing also prompts LPS rush p65 to shift into core, and Rosuvastatin can inhibit this existing As (Fig. 4).BMS-345541, is a kind of NF-kB signal pathway inhibitor, and Rosuvastatin shows similar effect (Fig. 4).

Rosuvastatin (10 μM) and (or) LPS (20ng/mL) and (or) BMS-345541 stimulation DPSCs after at dentine Induction 21 days.(A) Western Blot promotes p65 phosphorylation, I κ B α degradation and p65 nuclear transfer after analyzing LPS stimulation, and Rosuvastatin and BMS-345541 can inhibit this process；(B) in immuning fluorescent dyeing analysis DPSCs nucleus and cytoplasm P65 expression, core are dyed with DAPI, scale bar: 100 μm.

The above is only one embodiment of the present invention, it is noted that those skilled in the art, Without departing from the concept of the premise of the invention, some similar deformation and improvement can be made, these also should be regarded as this Within the protection scope of invention.

Claims (8)

1. purposes of the Rosuvastatin in the disease for the treatment of dentine or dentin matrix.

2. purposes according to claim 1, it is characterised in that: the disease includes saprodontia or hypoplasia.

3. purposes according to claim 1, it is characterised in that: Rosuvastatin is by promoting DPSCs to break up at dentine Treat the disease.

4. purposes according to claim 3, it is characterised in that: Rosuvastatin is in inflammatory microenvironment, by inhibiting NF- κ B signal access promotes DPSCs at disease described in dentine differentiation therapy.

5. purposes according to claim 1-4, it is characterised in that: Rosuvastatin first choice concentration is 1-30 μM.

6. purposes according to claim 5, it is characterised in that: Rosuvastatin first choice concentration is 5-25 μM.

7. purposes according to claim 6, it is characterised in that: Rosuvastatin first choice concentration is 8-20 μM.

8. purposes according to claim 7, it is characterised in that: Rosuvastatin first choice concentration is 10 μM.