CN104922108A - Application of parthenolide to medicine for inhibiting periodontal cell inflammation and bone destruction - Google Patents
Application of parthenolide to medicine for inhibiting periodontal cell inflammation and bone destruction Download PDFInfo
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Abstract
The invention discloses novel application of parthenolide medicine and mainly relates to the application of the parthenolide to a medicine for controlling periodontal ligament cell inflammation reaction and inhibiting bone destruction. Study shows that the parthenolide has an obvious effect in inhibiting periodontal cell inflammation reaction and bone destruction.
Description
Technical field
The present invention relates to field of medicaments, the particularly medicinal application of parthenolide in Periodontal ligament cellular inflammation reaction controlling and suppression bone destruction.
Background technology
It is the common problem of clinical oral that Tissues around tooth infects that the frontal resorption caused as the disease such as periapical periodontitis and periodontitis destroys, and severe patient can cause odontoseisis, being shifted even comes off.Periodontitis prevalence is up to 90% in adult, and the periodontal tissue defect that periodontitis causes is the main cause causing person in middle and old age's odontoseisis He come off.Clinical conventional periodontal subgingival debridement and root planing effectively can remove pathogenic microorganism, but how effectively to control inflammation, and making the Tissues around tooth physiological regeneration destroyed be the target that clinical oral is treated, is also key and the difficult point of periodontal treatment.Tissues around tooth inflammation often involves the tissues such as cementum, periodontal membrane and alveolar bone.The formation of alveolar bone and absorption are the dynamic equilibrium of skeletonization and broken bone function.Alveolar bone cell plays an important role in maintenance osteogenesis with the Periodontal ligament cell with differentiation potential, and osteoclast and precursor macrophage thereof play the effect of bone resorption and dissolving, and both mutually work in coordination with under physiological status, complete bone formation and reinvent.
The periodontal local application of current listing, based on antibiotics, easily causes the adverse consequencess such as bacterial drug resistance.And periodontitis is after the treatment such as periodontal scaling and root planing, and most patient has reached the effect controlling bacteriological infection, does not need the auxiliary treatment of local antibiotic class medicine.But inflammation causes bone destruction and the new bone formation mechanism of being obstructed to be focus and the difficult points of Recent study.Non-steroidal anti-inflammatory drugs (NSAIDs) uses maximum antiinflammation pain-stopping pharmaceuticals clinically at present, and its mechanism of action suppresses the synthesis of epoxide hydrolase (COX-2) and prostaglandin.But continuing of periodontal tissue's inflammation is the comprehensive function result of the inflammation factor, relates to Periodontal ligament cell, osteoblast, the normal physiological function of osteoclast and precursor macrophage thereof simultaneously.Research macrophage, osteoclast and osteoblast participate in inflammatory bone resorption and generting machanism, find new Drug therapy action target spot, the medicine of regulation and control inflammatory signals path is utilized effectively to control the degraded of diseased region alveolar bone, promote the orderly regeneration of alveolar bone, periodontal osseous tissue and periapical tissue, there is important clinical meaning.
Research shows, nuclear factor (nuclear factor kappa b, NF-κ B) signal path is not only the key factor of regulation and control inflammatory reaction gene expression, is also the ripe important regulatory factor with affecting cell Osteoblast Differentiation of regulation and control differentiation of osteoclast.Parthenolide (Parthenolide, PTL) is the natural product class medicine of specific inhibition NF-κ B signal path.PTL is a kind of natural sesquiterpene alkene lactone medicine, has analgesia, antibacterial and antitumor action, is commonly used to the diseases such as treatment heating, migraine and arthritis.Experiment in vitro confirms that PTL has the effect suppressing the synthesis of the inflammation factor and release, by IL-1, IL-6, TNF-α, IL-8, and prostaglandin, the suppression of the cytokines such as COX2 and nitric oxide synthetase (NOS) and play antiinflammatory action.Zoopery confirms that PTL is by suppressing the activity of NF-κ B, reduces the content of IL-6 and TNF in blood circulation, thus the inflammatory reaction that blocking-up lipopolysaccharide (LPS) is induced.In addition, in the research of macrophage and osteoclast, find the NF-kB activation that PTL can suppress LPS to induce, p65 nuclear translocation and I-κ B degrade.More than study prompting, the pharmacologically active of PTL antiinflammatory realizes by suppressing NF-κ B signal path.
Summary of the invention
Research purpose of the present invention is a kind of dentistry topical drugs suppressing the reaction of Periodontal ligament cellular inflammation and suppress periodontal tissue's bone destruction of research and development.This medicine uses as the ancillary drug treatment after periodontal scaling and operative treatment clinically, thus reaches the object promoting that periodontal tissue repairs and regenerates.
Parthenolide, as a kind of active constituents of medicine, can extract by extracting method conveniently in Sesquiterpene class plant.Inventor studies discovery by experiment, PTL is suitable for the concentration range (less than or equal to 5 μMs) of Periodontal Ligament Cells growth, in the inflammatory reaction of bacterial endotoxin (LPS) in-vitro simulated periodontal, the application of PTL can significantly reduce inflammatory factor IL-1 in Periodontal Ligament Cells, the gene expression of IL-6 and TNF-α, inhibit osteoclast cell activation related gene RANKL and M-CSF simultaneously, raise the expression of the antagonizes factor OPG of RANKL.And PTL is by suppressing the expression of p50 and suppressing I-κ B degrade thus suppress the activation of NF-κ B signal path in Periodontal Ligament Cells.More than research display parthenolide has the effect suppressing the reaction of Periodontal ligament cellular inflammation.
The reparation of tooth body surrounding bone tissue depends on the Periodontal ligament cell with differentiation potential, and osteoclast and precursor macrophage thereof play the effect of bone formation and dissolving.Both mutually work in coordination with under physiological status, complete bone formation and reinvent.Inventor builds the model of Periodontal ligament cell and macrophage Dual culture, research finds that PTL inhibits macrophage ripe to differentiation of osteoclast by experiment, and lower the key gene that broken bone function is relevant, as RANK, Calcitonin Receptor, Carbonic Anhydrase II, MMP-9, Cathepsin K and TRAP.Above result display PTL has the differentiation suppressing osteoclast and the function suppressing bone destruction.
The pharmacodynamic experiment of the present inventor shows, parthenolide has remarkable inhibitory action to the reaction of Periodontal ligament cellular inflammation and bone destruction model, and embodiment 1 is shown in concrete analysis.
As beneficial effect of the present invention:
Parthenolide PTL has the effect suppressing periodontal tissue's inflammatory reaction, suppresses the differentiation of osteoclast and broken bone function simultaneously, has the effect suppressing bone destruction, and its mechanism of action realizes by suppressing NF-κ B signal path.
Parthenolide has potential therapeutic value to periodontitis.
As the antiinflammatory of targeted inhibition NF-κ B signal path of new generation and the natural product class medicine of anti-bone destruction, there is wide potential applicability in clinical practice.
Parthenolide PTL can be made into PTL sustained-release gel, as the ancillary drug treatment after periodontal treatment, is placed in periodontal pocket and periodontal affected part, reaches the object promoting periodontal tissue's reparation and regeneration.
Accompanying drawing explanation
Fig. 1 is that parthenolide (PTL) is on the schematic diagram of the impact of PDLCs ability of cell proliferation;
Fig. 2 is that parthenolide (PTL) is on the schematic diagram of the impact of PDLCs cell-signaling pathways;
Fig. 3 is that parthenolide (PTL) breaks on PDLCs cellular inflammation Summing Factor the schematic diagram that bone photo closes the impact that factor gene is expressed;
Fig. 4 is that parthenolide (PTL) is on the schematic diagram of macrophage to the impact (TRAP coloration result) of differentiation of osteoclast;
Fig. 5 is the schematic diagram of parthenolide (PTL) on the impact of bone function related gene broken in osteoclast.
Detailed description of the invention
Now 1 to 5 illustrate that the present invention is further described with embodiment by reference to the accompanying drawings:
Embodiment 1 pharmacodynamics test: parthenolide is to periodontal cell inflammatory reaction and bone destruction Inhibition test:
Specific experiment process is as follows:
1, material:
1). Periodontal Ligament Cells (PDLCs) and macrophage:
Collect The Stomatologial Hospital of Zhongshan University Oral and Maxillofacial Surgery out-patient (18 ~ 25 years old) pull out because of impaction or orthodontic treatment health, complete, without dental caries permanent teeth (after patient's informed consent).Scraping periodontal tissue carries out the original cuiture of Periodontal Ligament Cells.
Adopt macrophage Macrophage, namely RAW264.7 cell line is as osteoclast precursor, for this experiment.
2). medicine and reagent:
1.. parthenolide (PTL), experiment reagent, purchased from American Sigma company.The storage liquid being mixed with 20mmol/L is dissolved with 100%DMSO; Desired concn is diluted to, i.e. 1,5,10 and 20 μMs with the DMEM culture medium (dual anti-containing mycillin) containing 10% hyclone during use.
2.. gram negative bacteria induced by lipopolysaccharide (LPS, E.coli055:B5), purchased from American Sigma company, dissolves preparation storage liquid with PBS solution; 1 μ g/mL is diluted to the DMEM culture medium (dual anti-containing mycillin) containing 10% hyclone during use.
3.. the low sugar culture-medium of cell culture fluid: DMEM, hyclone (FBS) purchased from American Gibco company; Mycillin dual anti-purchased from American Sigma company.
4.. TRIzol Reagent: extract mRNA cell cracking agent, purchased from Australian Life Technologies company.
5.. DyNAmo cDNA Synthesis Kit: Reverse Transcriptase kit, purchased from Australian Life Technologies company.
6.. SYBR reagent:QRT-PCR reaction reagent, purchased from American ABI company.
7.. TRAP staining assay kit:TRAP staining kit, for the dyeing of multinucleated osteoclast, purchased from American Sigma company.
3). experimental apparatus:
1.: ABI 7500 Thermal Cycler:QRT-PCR instrument, Australian Applied Biosystems company.
2.: microplate reader: Molecular Devices company of the U.S., SpectraMax Microplate Reader series.
3.: inverted phase contrast microscope: Nikon company, Nikon ECLIPSE, TS100 series.
2, experimental technique:
1). the cultivation of Periodontal Ligament Cells and macrophage:
After collecting tissue of tooth sample, for subsequent use after rinsing 3 times with the aseptic PBS containing 5% dual anti-(green grass or young crops/streptomycin); Use the periodontal membrane tissue of in the middle part of knife blade scraping root of the tooth 1/3 in super-clean bench, be cut into about lmm with eye scissors
3the piece of tissue of size; Collection organization's block, is inoculated in 25cm by piece of tissue
2in culture bottle, put 37 DEG C, cultivate in 5%CO2 incubator.After 7-8h after piece of tissue major part is adherent, add the DMEM culture medium (containing mycillin dual anti-) of 3 mL containing 10%FBS, within 2-3 days, observe and change liquid 1 time.Observation of cell upgrowth situation under inverted phase contrast microscope, when Growth of Cells reaches 80% fusion, adopts trypsin digestion (0.25% pancreatin+0.02%EDTA) to carry out cell l:3 and go down to posterity, get 3rd ~ 6 generation cell carry out subsequent experimental.
Macrophage system RAW264.7 is put 37 DEG C, 5%CO
2cultivate in incubator, cell culture medium is the DMEM culture medium (dual anti-containing mycillin) containing 10%FBS, within 2-3 days, observes and changes liquid 1 time.
2). mtt assay detects parthenolide (PTL) to the impact of PDLCs multiplication capacity:
Get well-grown 3rd ~ 5 generation PDLCs, with 3 × 10 after digestion
3individual/mL density is inoculated in 9 piece of 96 orifice plate, every block plate 30 hole, and every hole 100 μ L, at 37 DEG C, 5%CO
2incubator is cultivated cellar culture 12h and is ensured cell attachment growth.Empirically divide into groups replaced medium, and PTL concentration is respectively 0,1,5,10 and 20 μM; Continue cultivation after 1,3 and 7 day, every hole adds 10 μ L MTT reagent, 37 DEG C hatch 4h after, dissolve the formazan generated with DMSO.Use microplate reader 495nm to add a cover and measure each hole OD value, carry out statistical analysis.
3). parthenolide (PTL) is on the impact of NF-κ B signal path in PDLCs cell:
This experiment adopts Western blot to detect parthenolide (PTL) to the impact of NF-κ B signal path in PDLCs cell.
Get well-grown PDLCs cell, with 1 × 10 after digestion
5individual/mL density is inoculated in 6 orifice plates, every hole 1.5 mL cell suspension, 37 DEG C, 5%CO
2incubator cellar culture 24h ensures cell attachment growth; With 1 μM of PTL pretreatment cell 1h, then with 1 μ g/mL Escherichia coli LPS irritation cell; After LPS adds, at 15,30 and 60 minutes point collecting cell whole proteins; Lysis formula of liquid is: 1% Ipegal, 1% sodium dodecyl sulphate, 50 mM Tris-HCl and 150 mM NaCl, freshly before lysis adds protease inhibitor (Roche, Swiss); Protein concentration is measured with BCA method.
Join 10%SDS-PAGE separation gel and 4% concentrated glue carry out gel electrophoresis.
Albumen sample applied sample amount is 10 μ g/ holes, after electrophoretic separation, goes to NC film; After closing, hatch primary antibodie for 4 DEG C and spend the night.Primary antibodie comprises: I-κ B, NF-κ B, ERK and p-ERK (1:1000), equal purchased from American Cell Signalling Technology company.Internal reference antibody GAPDH (1:1000) is purchased from Abcam.Then, add corresponding fluorescence two and resist, after hatching 1h, scan band brightness; Measure gray value, carry out graphical analysis.
4). parthenolide is to the Inhibition test of periodontal cell inflammatory reaction and broken bone function:
1. parthenolide breaks on Periodontal ligament cellular inflammation Summing Factor the impact that bone photo closes factor expression:
This experiment adopts QRT-PCR detection parthenolide (PTL) to close the impact of factor expression to inflammatory factor in PDLCs cell and broken bone photo.
Get well-grown PDLCs cell, with 1 × 10 after digestion
5individual/mL density is inoculated in 6 orifice plates, every hole 1.5 mL cell suspension, 37 DEG C, 5%CO
2incubator cellar culture 24h ensures cell attachment growth.With 1 μM of PTL pretreatment cell 1h, then with 1 μ g/mL Escherichia coli LPS irritation cell.After LPS adds, collect sample at 1 and 3 day time point.Utilize TRIzol cell lysis, extract mRNA.With DyNAmo cDNA Synthesis Kit Reverse Transcriptase kit synthesis cDNA.
By the sequence obtaining people's inflammation-related gene (IL-1, IL-6 and TNF-α) and broken bone function related gene (RANKL, OPG and M-CSF) in Gene Bank data base, design and synthesis primer sequence.Primer sequence is in this experiment of table 1. QRT-PCR primer sequence used.Adding reaction system in every sample utilizes full-automatic quantitative real time PCR Instrument to carry out PCR reaction with SYBR Green dye method.After reaction terminates, by computer automatic analysis fluorescence signal, collect fluorescence data, calculate the horizontal relative quantity of mRNA of each sample genes of interest and reference gene, carry out statistical analysis.
2. Periodontal ligament cell (PDLCs) and macrophage (Macrophage) Dual culture:
This experiment adopts PDLCs and osteoclast precursor-macrophage (Macrophage) co-culture system, and whether study parthenolide (PTL) affects PDLC to the regulating action of macrophage to differentiation of osteoclast and broken bone function.
Adopt 12 hole Transwell cells (pore size is 0.5 μm), upper room inoculation PDLCs, adds the PTL(0 containing variable concentrations, 1 and 5 μM) DMEM culture fluid, and with endotoxin LPS irritation cell, matched group is DMEM culture fluid group, after stimulating 48h, discard culture fluid.Inoculation macrophage in room under Transwell, cell culture fluid is the DMEM culture fluid containing 10 ng/mL RANKL.By PDLCs and macrophage Dual culture, after 7 days, collection of cellular samples, detect osteoclast related gene (RANK, Calcitonin Receptor, Carbonic Anhydrase II, MMP-9, Cathepsin K and TRAP) expression in macrophage, the RT-PCR method adopted is closed described in the impact of factor expression as parthenolide in the present invention breaks bone photo to Periodontal ligament cellular inflammation Summing Factor; Primer sequence is in this experiment of table 1. QRT-PCR primer sequence used.
The primer sequence that this experiment of table 1. QRT-PCR is used
3. TRAP dyeing:
By the method for following Periodontal ligament cell (PDLCs) and macrophage (Macrophage) Dual culture by PDLCs and macrophage Dual culture after 7 days, carry out TRAP dyeing and observe macrophage to the form of differentiation of osteoclast and quantity.
Concrete colouring method is as follows: fixed by macrophage RAW264.7, and shown fixative formula is as follows: 6.75 mM citrate, 65% acetone and 3.7% formalin.TRAP dyeing adopts Sigma TRAP staining test kit.The apocyte (three cores or more) of TRAP stained positive is osteoclast.Observe under optics inverted phase contrast microscope (Nikon ECLIPSE, TS100) and take pictures.
4. statistical analysis:
People's Periodontal ligament cell in this experiment employing three Different Individual sources, independently in triplicate.Data represent with mean ± standard deviation (Mean ± SEM), adopt SPSS18.0 software analysis.Comparison between many groups adopts one factor analysis of variance (one way analysis of variance, ANOVA), if variance is neat, difference has significance, compares and adopt S-N-K inspection between group.Think that difference has statistical significance with p< 0.05.
3, experimental result:
1). PTL is on the impact of PDLCs ability of cell proliferation:
As shown in Fig. 1 or lower chart, MTT cell proliferation experiment result shows, concentration is that 1,5 and 10 μM of PTL acts on PDLCs after 1 day and 3 days, and each experimental group OD value compares with matched group, no significant difference (p>0.05).The OD value of 20 μMs of PTL experimental grouies is starkly lower than matched group, and difference has statistical significance (p<0.05), and namely on cell proliferation has significant inhibitory action.This result show lower concentration PTL(1 and 5 μM) PDLCs propagation is had no significant effect, high concentration is antiproliferative effect then.
PTL is on the impact of PDLCs ability of cell proliferation
* compare with matched group, p < 0.05;
2). parthenolide (PTL) is on the impact of NF-κ B signal path in PDLCs cell:
NF-κ B and ERK signal path play an important role in periodontal disease reaction, and this experiment adopts western blot to detect the impact of PTL on the middle NF-κ B of Periodontal ligament cell (PDLCs) and ERK signal path activation levels.As shown in Fig. 2 or lower chart, endotoxin LPS stimulates PDLCs cell, significantly can raise NF-κ B/p50 and p-ERK protein level, and present remarkable downward trend as the I-κ B of NF-κ B Profilin.But at 30 and 60 minutes points, compare with LPS experimental group, PTL obviously can reduce NF-κ B/p50 and p-ERK protein level, raise the protein level of I-κ B.Above result display PTL suppresses the activation of ERK and NF-κ B signal path.
Parthenolide (PTL) is on the impact of PDLCs cell-signaling pathways
# compares with blank group (Control), p < 0.05; * compare with LPS group, p < 0.05;
3). parthenolide (PTL) closes the impact of factor gene expression to Periodontal ligament cell (PDLCs) inflammatory factor and broken bone photo:
According to MT reconnaissance T cell proliferation experiment result, this experiment adopts the PTL concentration not affecting cell proliferation, and namely concentration is the PTL of 1 and 5 mM.As shown in Fig. 3 or lower chart, at 1 day and 3 days time points, LPS significantly raised the gene level of surveyed inflammatory factor and broken bone correlation factor.But compare with LPS experimental group, at 1 day and 3 days time points, 5 μMs of PTL obviously can reduce IL-1, the gene expression dose of IL-6, TNF-α, RANKL and M-CSF.At 3 days time points, 1 μM of PTL also obviously can reduce the gene level of the surveyed factor.The gene activation of the inflammatory factor that above result display PTL suppresses LPS to cause and broken bone correlation factor.
PTL breaks on PDLCs cellular inflammation Summing Factor the impact that bone photo closes factor gene expression
(A) expression of inflammatory factor (IL-1 β, IL-6 and TNF-α) gene; (B) expression of broken bone correlation factor (M-CSF, RANKL and OPG) gene.# compares with blank group (Control), p < 0.05; * compare with LPS group, p < 0.05;
4). parthenolide (PTL) is on the impact of macrophage to differentiation of osteoclast:
As described in above-mentioned experimental technique, by macrophage and PDLCs Dual culture after 7 days, the differentiation situation of osteoclast is observed in TRAP dyeing.A large amount of macrophage RAW264.7 can be induced to differentiation of osteoclast with LPS pretreated PDLCs cell.But, with PTL pretreatment PDLCs cell, the differentiation of osteoclast maturation that LPS pretreatment causes significantly can be reduced.This result display PTL suppresses macrophage to the differentiation of osteoclast.As shown in Figure 4, Experimental comparison results is as follows:
Parthenolide (PTL) is on the impact (TRAP dyeing) of macrophage to differentiation of osteoclast
# compares with blank group (Control),
p< 0.05; * compare with LPS group,
p< 0.05.
5). parthenolide (PTL) is on the impact of bone function related gene broken in osteoclast:
As described in above-mentioned experimental technique, by macrophage and PDLCs Dual culture after 7 days, detect PTL to bone function related gene (RANK broken in osteoclast, Calcitonin Receptor, Carbonic Anhydrase II, MMP-9, Cathepsin K and TRAP) impact expressed.As shown in Fig. 5 or lower chart, the high expressed (p < 0.05) of institute's cls gene in osteoclast can be induced with the pretreated PDLCs cell of LPS.But, with 5 μMs of PTL pretreatment PDLCs cells, the rise (p < 0.05) of the broken bone function related gene that LPS pretreatment causes significantly can be reduced.This experimental result further illustrates the inhibitory action of PTL to bone function broken in osteoclast.
Parthenolide (PTL) is on the impact of bone function related gene broken in osteoclast
# compares with blank group (Control), p < 0.05; * compare with LPS group, p < 0.05;
4, conclusion:
The result of above-mentioned experiment shows, parthenolide has significant effect to suppression periodontal cell inflammatory reaction and bone destruction.
Embodiment 2, gel:
Parthenolide is mixed with proper auxiliary materials, makes gel.
Formula:
Parthenolide 5mg;
Gel 100mL;
Make parthenolide gel 100 mL (50 μ g/mL).
The invention of this project has the dentistry local PTL medicine of antiinflammatory and anti-osteoclasia.Can be made into PTL sustained-release gel, as the ancillary drug treatment after periodontal treatment, be placed in periodontal pocket and periodontal affected part, reach the object promoting periodontal tissue's reparation and regeneration.
Embodiment 3, gel:
Parthenolide is mixed with proper auxiliary materials, makes gel.
Formula:
Parthenolide 2.5mg;
Gel 100mL;
Make parthenolide gel 100 mL (25 μ g/mL).
The invention of this project has the dentistry local PTL medicine of antiinflammatory and anti-osteoclasia.Can be made into PTL sustained-release gel, as the ancillary drug treatment after periodontal treatment, be placed in periodontal pocket and periodontal affected part, reach the object promoting periodontal tissue's reparation and regeneration.
In sum, after those of ordinary skill in the art reads file of the present invention, make other various corresponding conversion scheme according to technical scheme of the present invention and technical conceive without the need to creative mental work, all belong to the scope that the present invention protects.
Claims (2)
1. parthenolide is suppressing the application in periodontal cell inflammation and bone destruction medicine.
2. parthenolide according to claim 1 is suppressing the application in periodontal cell inflammation and bone destruction medicine, it is characterized in that: described medicine is gel.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108434133A (en) * | 2018-06-11 | 2018-08-24 | 江苏食品药品职业技术学院 | A kind of parithenolide is used as the biomedical uses of 9 inhibitor of Bone Morphogenetic Protein |
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| CN108434133A (en) * | 2018-06-11 | 2018-08-24 | 江苏食品药品职业技术学院 | A kind of parithenolide is used as the biomedical uses of 9 inhibitor of Bone Morphogenetic Protein |
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Application publication date: 20150923 |