CN105079018A - Application of aesculin in preparing medicine capable of preventing and treating osteoporosis - Google Patents

Abstract

The invention belongs to the field of medicine, and specifically relates to an application of aesculin and a composition thereof in preparing a medicine capable of preventing and treating osteoporosis. The study shows that the traditional Chinese medicine aesculin can inhibit NF-kB transcription activation effect induced by RANKL, inhibit the formation of osteoclasts, and reduce the expression of osteoclast differentiation specific genes, and thus the traditional Chinese medicine aesculin has the prospect of being developed into the medicine capable of preventing and treating osteoporosis.

Description

The application of aseculin in preparation control medicine for treating osteoporosis

Technical field:

The invention belongs to field of medicaments, be specifically related to Cortex Fraxini effective ingredient and preventing and treating the application in osteoporosis.

Background technology:

Osteoporosis is a kind of with bone amount minimizing, and bone micro-structure destruction, causes bone fragility to increase, and the general metabolism osteopathia that fracture is feature easily occurs.Osteoporosis can betide any crowd and any age, very common in postmenopausal women and old people, is the key factor threatening senior health and fitness and reduce quality of life.

Osteoporosis become the current whole world be only second to cardiovascular disease, be injured crowd at most, most hazardness and not yet curable chronic disease.Estimate according to World Health Organization (WHO), global patient can reach 200,000,000 examples, and its sickness rate has leapt to the 7th of the various commonly encountered diseases in the world, and the physical and mental health of the mankind in serious threat, brings heavy family, society and economy burden.

Bone metabolism balance is that bone resorption two dynamic processes of bone formation and the Osteoclasts mediate mediated by osteoblast regulate and control exquisitely.When bone resorption exceedes bone formation, there will be bone amount and reduce, bone loss can occur, and osteoporosis.Therefore, anyly can directly or indirectly act on osteoblast and/or osteoclast, contribute to the material keeping its metabolism dynamic equilibrium, all likely there is the osteoporotic effect of control.

Osteoclast derives from the absorbent apocyte of monocytes/macrophages cording [TeitelbaumSL.Science, 2000,289:1504 – 8.].Ripe osteoclast has different kinds of molecules label, comprises Tartrate resistant acid phosphatase (TRAP), cathepsin K (CathepsinK), empty broken proton v-ATP (v-ATPased2) etc.Due to osteoclast energy tartrate resistant acid phosphatase secretion, TRAP stained positive, so TRAP stained positive can be used as the specific index of osteoclasts cultured in vitro most.

RANKL has by osteoblast/a kind of of stromal cell synthesis the cytokine stimulating colony stimulating factor group to be divided into osteoclast effect.After the TRAP in RANKL and downstream gathers, NF-κ B can be activated, thus cause brokenly the expression of bone related genes.NF-κ B participates in differentiation of osteoclast and ripe transcription factors critical [ChaissonMLetal.JBiolChem, 2004,279:54841-48].

Matrix metalloproteinase (MMP), belongs to zinc-binding protein endopeptidase families, and in order to comprise, to be organized in interior Various Tissues extracellular mechanisms of degradation necessary.MMP, especially MMP9 high expressed in osteoclast is degradation of cell epimatrix, participates in the important protease of bone remoulding, plays an important role in the bone resorption that broken bone active increases.

Osteoporotic Drug therapy becomes world subject.The osteoporotic drug main of current clinical treatment will be divided into: promoting bone growing medicine, suppress bone resorption medicine and improve 3 types such as osseous tissue medicine.

Promoting bone growing medicine mainly contains: fluoride, parathyroid hormone and similar compound, vitamin D and derivant, somatomedin, statins, prostaglandin E 2 medicine, strontium salt etc.;

Bone resorption medicine is suppressed to mainly contain: estrogens, calcitonin, bisphosphonates etc.;

Improve osseous tissue medicine medicine to mainly contain: calcium preparation, protein, vitamin C etc.

In addition, because Chinese medicine has the advantages such as overall medical treatment, good effect, toxic and side effects are little, how to utilize modernization of Chinese medicine technology from Chinese medicine, find the osteoporosis therapy medicine of new high-efficiency low-toxicity, become the important directions for the treatment of osteoporosis agents research and development at present.Conventional Chinese medicine mainly contains Herba Epimedii, Radix Rehmanniae Preparata, the Cortex Eucommiae, the Radix Astragali, Fructus Psoraleae, Radix Angelicae Sinensis, Rhizoma Drynariae, Carapax Et Plastrum Testudinis, Rhizoma Dioscoreae, Radix Salviae Miltiorrhizae, Poria, Semen Cuscutae, Colla cornus cervi, Fructus Corni, Herba Cistanches, Fructus Lycii etc.Zoopery and clinical experiment all show, and by the osteoporotic effective ingredient of pharmacological evaluation (comprising high flux screening) screening treatment from Chinese medicine, have huge potentiality to be exploited.

Cortex Fraxini is dry branch skin or the trunk bark of five kind of plant such as Oleaceae plants Chinese ash FraxinuschinensisRoxb, is China's conventional Chinese medicine simply, and begin to be loaded in the pharmacy monograph Shennong's Herbal of Han dynasty, and history tree is all on the books.Cortex Fraxini has effect of heat clearing and damp drying, astringent therapy, improving eyesight, is usually used in treatment hematodiarrhoea, has loose bowels, the disease such as the raw nebula film of leucorrhea with red and white discharge, conjunctival congestion and swelling pain, order.Modern pharmacological research shows, Cortex Fraxini has anti-inflammatory and antalgic, reduces hematuria acid, anticoagulant, eliminating phlegm and stopping cough are relievingd asthma and antitumor action, is mainly used in treating enteritis, dysentery, leucorrhea, chronic tracheitis, conjunctivitis, is used for the treatment of gout effect excellent.In certified products Cortex Fraxini, main component is Coumarins (aseculin, aesculetin, fraxin, fraxetin, place post white beeswax glycosides, 6,7-dimethoxy coumarin etc., in addition also have phenols, saponin and tannin etc., wherein aseculin, aesculetin are the active ingredients for the treatment of tracheitis and bacillary dysentery illness.But relating to aseculin of the present invention preventing and treating in osteoporosis applies, and up to now, there is not yet relevant report both domestic and external.

The object of the invention is, the application of aseculin in preparation control medicine for treating osteoporosis is provided.

Summary of the invention:

In order to provide aseculin preventing and treating the application in osteoporosis, the present invention takes following technical scheme:

Adopt the conventional cell model mononuclear phagocyte RAW264.7 cell of research osteoclast extracorporeal biology behavior, this cell can to differentiation of osteoclast under nuclear factor receptor activation factor ligand (RANKL) stimulates induction.

1, detect aseculin and suppress RANKL to induce the transcription activating effect of NF-κ B;

2, detect the formation effect that aseculin suppresses multinuclear mature osteoclast;

3, detect the gene expression that aseculin suppresses differentiation of osteoclast gene TRAP, MMP9, CathepsinK, v-ATPased2.

invention effect:

Beneficial effect of the present invention is, disclose the application of aseculin in the osteoporotic medicine of preparation control, in effective dose, obvious cytotoxicity be there is no to RAW264.7 cell, the NF-κ B transcription activating effect that RANKL can be suppressed to induce, suppresses the formation of RAW264.7 cell to osteoclast of RANKL induction.Meanwhile, the expression of the differentiation of osteoclast specific genes such as TRAP, MMP9, CathepsinK, v-ATPased2 can be reduced, suppress bone resorption, finally play the osteoporotic effect of control.Because aseculin is pure natural substance, drug safety, toxic and side effects is little, can be used for osteoporotic control.

Accompanying drawing illustrates:

Fig. 1: aseculin is to the cytotoxicity of RAW264.7 cell

Fig. 2: aseculin is on the impact of the NF-κ B transcriptional activity effect that RANKL induces.

Wherein: the dosage of the aseculin of abscissa-variable concentrations; The NF-κ B transcriptional activity that vertical coordinate-each group fluorescent value percent compared with blank group fluorescent value is stated

Fig. 3: aseculin suppresses osteoclast formation to RANKL

Fig. 4: aseculin suppresses the mrna expression of differentiation TRAP, CathepsinK, MMP9, v-ATPased2 in osteoclast

embodiment:

Following examples are only and help those skilled in the art to understand the present invention better, but do not limit the present invention in any way.The experimental technique of actual conditions is not indicated, usually the conveniently conditioned disjunction condition of advising according to manufacturer in embodiment.

" embodiment 1 " aseculin is to the cytotoxicity of RAW264.7 cell

By RAW264.7 cell (purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's Cell Culture Center), with 5 × 10 ³individual/well is inoculated in 96 orifice plates, every hole 100 μ L, after cell covers with, changing into containing final concentration is 0 (blank group), 0.1 μM, 1 μM, 10 μMs, the DMEM high glucose medium (containing 10% hyclone) of the aseculin (purchased from lark prestige Science and Technology Ltd.) of 30 μMs, continue cultivation 24 hours, before cultivation terminates 4 hours, every hole adds 20 μ LMTT (5mg/ml), supernatant is abandoned after cultivation terminates, every hole adds 150 μ LDMSO, concussion makes purple crystal granule dissolve completely, absorbance is measured in 570nm place, result as shown in Figure 1.As shown in Figure 1, in 0.1 ~ 30 μM of concentration range, aseculin does not have significant cytotoxicity to RAW264.7 cell.

" embodiment 2 " aseculin is on the impact of the NF-κ B transcriptional activity effect that RANKL induces

LipofectamineTMLTXandPLUSReagent transfection reagent box (invitrogen) is adopted plasmid pNF-κ B-Luc (Stratagene) with NF-κ B Reporter System.Concrete operations are with reference to description.

The cell of transfection pNF-κ B-Luc plasmid is with 1 × 10 ⁴individual/well kind is in 96 hole blanks, with empty plasmid (the green skies) for control plasmid, be placed in 37 DEG C of CO2 gas incubator, cultivate after 24 hours, after adding 50ng/mlRANKL (Peprotech) pretreatment 1h, add the aseculin effect 8 hours of variable concentrations, adopt fluorescence detection reagent kit (Promega) to carry out fluoroscopic examination.Operating procedure is as follows: remove supernatant in 96 orifice plates to be measured, clean 3 times with PBS.5 × cell pyrolysis liquid (CCLR) deionized water is diluted to 1 ×, add by every hole 20 μ l and treat in gaging hole.Be placed in 37 DEG C of incubators, cracking 30min.Take out ELISA Plate, add luciferase substrate solution 50 μ l, multiple labeling microwell plate detector detects immediately.As shown in Figure 2, aseculin can suppress the NF-κ B transcription activating effect of RANKL induction to result dose-dependant.

The impact that " embodiment 3 " aseculin induces TRAP positive cell (osteoclast) to produce on RANKL

By RAW264.7 cell with 2.5 × 10 ³individual/well is inoculated in 96 orifice plates, after cell covers with, discard culture medium, medium component: DMEM high glucose medium (containing 10% hyclone), then by following grouping inducing culture:

A:control; B:RANKL (50ng/ml) induces, without aseculin matched group; C:RANKL (50ng/ml) induces, 1 μM of aseculin experimental group; D:RANKL (50ng/ml) induces, 5 μMs of aseculin experimental grouies; E:RANKL (50ng/ml) induces, 10 μMs of aseculin experimental grouies; F:RANKL (50ng/ml) induces, 20 μMs of aseculin experimental grouies.

Every three days, change liquid once, inducing culture, after 6 days, carries out Tartrate resistant acid phosphatase (TRAP) dyeing, concrete with reference to sigma (386A) associative operation.At light Microscopic observation after dyeing terminates, as shown in Figure 3, aseculin can suppress the generation of osteoclast to result.

" embodiment 4 " aseculin suppresses the expression of differentiation of osteoclast specific gene

RAW264.7 cell is inoculated in 6 orifice plates with 300,000/well, culture medium is discarded after cell covers with, medium component: DMEM high glucose medium (containing 10% hyclone), then by following grouping inducing culture: group 1: without RANKL (50ng/ml) induction, without aseculin matched group; Group 2:RANKL (50ng/ml) induction, without aseculin matched group; Group 3:RANKL (50ng/ml) induction, 5 μMs of aseculin experimental grouies; Group 4:RANKL (50ng/ml) induction, 10 μMs of aseculin experimental grouies.

Every 3 days, change liquid once, after induction 5d, abandon culture medium, use Trizol cell lysis, extract total serum IgE in cell, after measuring RNA concentration and purity, carry out reverse transcription and prepare cDNA.Get 1 μ g total serum IgE and 4 μ l5 × PimeScriptRTMasterMix join in PCR pipe, and supply 20 μ l systems with the water without RNase.Reverse transcription condition: 37 DEG C of 30min, 85 DEG C of 5s, 4 DEG C of coolings.PCR adopts 20 μ l reaction system: 2 × EasyTaq, 10 μMs of PCR primer and 50ngcDNA template.Primer sequence is as follows: CathepsinK: forward primer: 5-cctctcttggtgtccataca-3 ' (SEQIDNO.1); Reverse primer: 5 '-atctctctgtaccctctgca-3 ' (SEQIDNO.2); TRAP: forward primer: 5 '-aaatcactctttaagaccag-3 ' (SEQIDNO.3); Reverse primer: 5 '-ttattgaatagcagtgacag-3 ' (SEQIDNO.4); MMP9: forward primer: 5 '-ctgtccagaccaagggtacagcct-3 ' (SEQIDNO.5); Reverse primer: 5 '-gtggtatagtgggacacatagtgg-3 ' (SEQIDNO.6); V-ATPased2: forward primer: 5 '-aagcctttgtttgacgctgt-3 ' (SEQIDNO.7); Reverse primer: 5 '-ttcgatgcctctgtgagatg-3 ' (SEQIDNO.8); GAPDH: forward primer: 5 '-catggccttccgtgttccta-3 ' (SEQIDNO.9); Reverse primer: 5 '-cctgcttcaccaccttcttgat-3 ' (SEQIDNO.10); Reaction condition: 94 DEG C of 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min.As shown in Figure 4, aseculin can suppress the expression of osteoclast specific gene TRAP, CathepsinK, MMP9, v-ATPased2 at 5,10 μMs to result concentration dependant.

Claims (2)

1. the application of aseculin in preparation control medicine for treating osteoporosis, it is characterized in that, described application system is based on suppressing aseculin RANKL to induce NF-κ B transcription activating effect, suppressing the formation effect of multinuclear mature osteoclast and suppress differentiation of osteoclast gene TRAP, MMP9, CathepsinK, v-ATPased2 to express.

2. be that the compositions that active component and one or more carriers pharmaceutically acceptable form is preparing the application prevented and treated in medicine for treating osteoporosis with aseculin.