CN108272795A - A kind of application of monascin in preparing treatment and delaying osteoarthritis drugs - Google Patents

Abstract

It is an object of the present invention to provide a kind of application of monascin in preparing treatment and delaying osteoarthritis lesion drug.The side effect larger compared to Western medicine, monascin, can more leniently relief from osteoarthritis lesions as a kind of food extracts.

Description

A kind of application of monascin in preparing treatment and delaying osteoarthritis drugs

Technical field

The present invention relates to a kind of application of monascin, more particularly to monascin is used to prepare treatment and delays Bones and joints Application in scorching drug.

Background technology

Osteoarthritis is most common knee joint degenerative disease, is apt to occur in 50 years old or more crowd.Clinical manifestation arthritis Bitterly, stiff, especially for a long time after activity, the quality of life of the different degrees of influence middle-older patient of this disease.But it is at present Only, clinical treatment is based on non-steroidal drug and replacement knee in arthroplasty.Non-steroidal drug can only improve the remission of disease Pain cannot reverse osteoarthritis progression, and there is also costly postoperative infection equivalent risks for knee prosthesis.

Osteoarthritis can be caused by many factors, such as abnormal stress, local nutritional deficiency and congenital gene unconventionality, Middle inflammation is one of most important pathogenic mechanism of osteoarthritis.By inflammatory factor mediate cartilage cell's dysfunction be considered as An important ring in osteoarthritis retrogression pathological changes process, therefore may for the research of inflammation mediated cartilage cell's dysfunction It is the important channel for treating osteoarthritis.

Red rice is product of the rice after Fermentation Condition of Monascus spp, has been had go through within more than 1000 years as food and nutriment History.Monascin (Monascin) is a kind of pigment extract of red rice.Monascin is shown in multinomial pharmaceutical research Good bioactivity, such as antitumor, immunosupress, septicopyemia disease etc..But its to osteoarthritis disorders whether It is unclear with therapeutic effect.

Invention content

It is an object of the present invention to provide a kind of application of monascin in preparing treatment and delaying osteoarthritis lesion drug. The side effect larger compared to Western medicine, monascin, can more leniently relief from osteoarthritis diseases as a kind of food extracts Become.

The technical solution adopted by the present invention is：

The present invention provides a kind of application of monascin in preparing treatment and delaying osteoarthritis drugs, the monascus Plain molecular structural formula is shown in formula (I)：

Further, the drug further includes pharmaceutical carrier, and the pharmaceutical carrier is micro emulsion, nano liposomes etc..

Further, the drug is tablet, emulsion or capsule etc..

Further, the drug is compound formulation, including erythromycin and is selected from polycaprolactone, the poly- second of polylactic acid-two Alcohol or polyvinyl alcohol contain material.

Further, the monascin dosage of the drug is 5-10mg/kg weight.

Compared with prior art, advantageous effect of the present invention is mainly reflected in：Monascin having good life as a kind of The active food extract of object has dosage low, the apparent feature of antiphlogistic effects.

Description of the drawings

Fig. 1：Therapeutic effect of the monascin to mouse medial meniscus of knee joint excision buckling form (DMM).Sham is Sham-operation group, DMM are osteoarthritis group, and DMM+Ms is osteoarthritis+monascin group.A：X-ray Imageology.B：SO is dyed (proteoglycans and glycosaminoglycan dyeing) Histological change.C：Histological score.

Fig. 2：The CCK8 toxicity trial compares figures of monascin.

Fig. 3：QPCR, ELISA and Western that monascin influences cartilage cell's inflammatory parameters in alleviation IL-1 β Blot experimental control figures；A is expressions of the iNOS and COX-2 in mRNA level in-site；B is TNF-α and IL-6 in mRNA level in-site Expression；C is expressions of the iNOS and COX-2 in protein level；D is iNOS and COX-2 gray value ratio statistical charts；E It is PGE2 and Nitrite in extracellular expression；F is TNF-α and IL-6 in extracellular expression.

Fig. 4：For cartilage cell under IL-1 β stimulations, monascin improves the compares figure of its extracellular matrix complex functionality；A For II Collagen Type VIs (Collagen II), proteoglycans (Aggrecan), MMP13, ADAMTS-5 protein level expression feelings Condition；B is Collagen II, Aggrecan, MMP13, ADAMTS-5 gray value ratio statistical chart；C is II Collagen Type VIs The expression fluorogram of (Collagen II) in protein level；D is II Collagen Type VIs (Collagen II) in the expression of protein level Fluorescent value ratio statistical chart.

Fig. 5：Cartilage cell is under IL-1 β stimulations, Western blot experimental control of the monascin to its NF- κ B access Figure；A is expression of the NF- κ B signals accesses in protein level；B is the cellular immunofluorescence experimental control that p65 enters core situation； C is the statistical chart of p65 and Lamin B gray value ratios；D is the statistical chart of I κ B α and GAPDH gray value ratios.

Specific implementation mode

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1

1, experimental procedure：

(1) modeling method：Take C57BL/6 Male wild-types (WT) mouse of 60 10 week old.Pass through medial meniscus (DMM) operation goes to stablize inducing mouse osteoarthritis.Peritoneal injection 2% (w/v) amobarbital (40mg/kg) is anaesthetized Afterwards, then iodophor disinfection cuts right capsula articularis genus on the inside of kneecap tendon, medial meniscus ligament is cut off with microsurgery scissors. Mouse is randomly divided into sham-operation group, physiological saline group and monascin treatment group.After DMM, monascin administration group (10mg/kg/ days；Continuous 8 weeks once a day by applying the monascin being dissolved in CMC in stomach).Meanwhile it is small in vehicle group Mouse is administered the CMC of equivalent.All animals are condemned to death after eight weeks after surgery, collect cartilaginous tissue sample and are penetrated for histology X Line analysis.

(2) Primary mouse cultured chondrocytes：With ten immature C57BL/6 mouse (5 of excessive yellow Jackets pair Only male and 5 females, 10 days) sentence euthanasia.The knee of mouse is aseptically carefully collected by disecting microscope Cartilage, and at 37 DEG C tissue is handled with 2mg/ml (0.1%) clostridiopetidase As II 4 hours.The cartilaginous tissue inoculation that digestion is suspended Into tissue culture medium.Cultured chondrocytes are containing 10% fetal calf serum (FBS；Hyclone, Thermo Scientifc, Logan, UT, USA) and 1% penicillin/streptomycin antibiotic (Gibco, Invitrogen, Grand) DMEM/F12 In (Gibco, Invitrogen, Grand Island, NY), 5% carbon dioxide, 37 DEG C of incubator.After cultivating 24 hours more Change culture medium.When up to 80% to 90% degree of cross linking, by using 0.25% trypsase-EDTA (Gibco, Invitrogen cell) is harvested.Then, cell is implanted into density appropriate in 10cm culture dishes again.Second generation cartilage is thin Born of the same parents are used for our all experiments, because passing on for 0 generation to significant variation is not observed during 2 generations.Cartilage cell is 5% CO₂, in culture in the incubator of 37 DEG C of conditions, complete medium is every other day replaced once.

(3) CCK8 methods measure drug cytotoxicity and drug antiphlogistic effects：CCK8 methods measure drug cytotoxicity：By Two generation cartilage cells are transferred in 96 orifice plates (50000cells/cm2), and in the monascin of various concentration (5,10,15,30 μ M it is incubated 24 hours and 48 hours in).At the appointed time, cell is washed with phosphate buffered saline (PBS), then contains 100 μ l There is the DMEM/F12 of 10 μ lCCK-8 to be added in each hole of tablet, and is incubated again 2 hours at a temperature of 37 DEG C.Then it uses The absorbance of spectrophotometer measured hole at 450nm.

(4) cartilage cell of step (2) is divided into 5 groups, and group 1,2 is control group (with 100% DMEM/F12 basal mediums For control), 35 μm of monascin+DMEM/F12 basal mediums of addition of group, 4 10 μm of monascin+DMEM/F12 bases of addition of group Basal culture medium, 4 15 μm of monascin+DMEM/F12 basal mediums of addition of group, after 37 DEG C of cultures for 24 hours, group 2, group 3, group 4 abandon training IL-1 β+DMEM/F12 the basal mediums of final concentration 10ng/ml are separately added into after foster base again, after 37 DEG C act on 24 hours, are collected Supernatant detects to the ELISA of inflammatory factor, and cell precipitation separation cell protein and cell RNA carry out Western respectively ELISA is used in combination to detect the expression situation of change of cartilage cell's its endocellular function albumen under inflammatory stimulus in blot and qPCR The case where detecting cell secretion inflammatory factor.

(5)Western blots：The cell precipitation RIPA method leach proteins after step (4) effect for 24 hours are taken, BCA methods are used in combination Survey albumen concentration.It takes protein extract that Loading buffer and water is added, makes the quantification of 30 μ g of protein content, 100 DEG C of heating 5min makes albuminous degeneration.The protein liquid after 20 μ l denaturation, PAGE gel electricity are added in each hole of PAGE gel Transferring film to pvdf membrane, 5% skimmed milk power room temperature closes 2h, primary antibody (primary antibody: primary antibody dilution volume ratio=1: 1000) 4 DEG C after swimming Overnight, TBST washs pvdf membrane 5min × 4 time, and secondary antibody (secondary antibody: volume ratio=1 TBST: 1000) is incubated at room temperature 2h, TBST washings 5min × 4 time, the development of ECL systems.GAPDH takes purpose band to inhale as internal reference, Image lab 3.0 pick-up slip band gray values Luminosity is with internal reference band ratio as relative expression quantity.Anti- p65, I κ B α primary antibodies are purchased in Cell Signaling Technology, Inc. (Beverly, MA, USA), anti-mm P-13, ADAMTS-5, iNOS, COX-2 primary antibody are purchased in Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-collagen-II and aggrecan primary antibodies are purchased in Abcam (Cambridge, MA, USA).Secondary antibody is purchased in Santa Cruz Biotechnology (Santa Cruz, CA, USA).

(6) real-time fluorescence quantitative PCR：It takes the cell precipitation after step (4) effect for 24 hours to carry RNA with Trizol methods, and measures Concentration.Reverse transcription is carried out with PrimeScriptTM Reverse Transcriptase kits, is tested with SYBR (Shanghai life work) kit, and And statistical data, carry out differential expression analysis.

Target gene primer sequence：

COX-2 (F), SEQ ID NO.1：5’-TCCTCACATCCCTGAGAACC-3’；

COX-2 (R), SEQ ID NO.2：5’-GTCGCACACTCTGTTGTGCT-3’；

INOS (F), SEQ ID NO.3：5’-GACGAGACGGATAGGCAGAG-3’；

INOS (R), SEQ ID NO.4：5’-CACATGCAAGGAAGGGAACT-3’；

IL-6 (F), SEQ ID NO.5：5’-CCGGAGAGGAGACTTCACAG-3’；

IL-6 (R), SEQ ID NO.6：5’-TCCACGATTTCCCAGAGAAC-3’；

TNF-α (F), SEQ ID NO.7：5’-ACGGCATGGATCTCAAAGAC-3’；

TNF-α (R), SEQ ID NO.8：5’-GTGGGTGAGGAGCACGTAGT-3’.

(7) ELISA detects the cellular inflammation factor：Step (4) is taken to act on the supernatant obtained afterwards for 24 hours, according to kit (R＆ D Systems, Inc., Minneapolis, MN, USA) operating procedure addition reagent, it is used in combination microplate reader to detect absorbance.

(8) cellular immunofluorescence：Cell precipitation after step (4) effect for 24 hours is seeded in 6 orifice plates slide, passes through cell Immunofluorescence test each group (packet synchronization is rapid (the 4)) expression of collagen II and the intracellular targeting situation of p65.With PBS is washed 5 minutes × 3 times, then is fixed with 4% paraformaldehyde.0.5% TRITON is added after washing 5min × 3 time with PBS X-100 handles 10min.5min × 3 time are washed with PBS, then 30min is closed with 5% BSA.Blot PBS, be added dropwise p65 (1: 200, CST) and II Collagen Type VIs antibody (1: 200, Abcam), 4 DEG C overnight.Next day is used after PBS washs 5min × 3 time with cell Green fluorescence secondary antibody (1: 400, Bioworld) room temperature, which is protected from light, is incubated 1h.After being washed with PBS, then with DAPI contaminate core.It is washed again with PBS After washing 5min × 3 time, anti-fluorescence quenching, mounting is added dropwise.Slide is taken in fluorescence microscopy microscopic observation and is shot.

2, experimental result：

(1) x-ray of simple Bones and joints group (DMM in Fig. 1) shows that joint space obviously narrows, articular surface calcification.And it is red After aspergillus extract for treating (DMM+Ms in Fig. 1), articular surface and joint space improve significantly relative to DMM groups, it was demonstrated that red yeast rice Mycin can significantly delay the progress (Fig. 1) of osteoarthritis.

(2) after obtaining absorbance by CCK8 experiments, we count the ratio of each group absorbance/control group absorbance, knot Fruit shows under 5,10,15,30 μm of monascin effects that interior cartilage cell's light absorption value and 0nN groups (no monascin group) nothing are aobvious for 24 hours Significant difference is write, shows 5,10,15,30 μm of monascins to cell without notable toxicity；And 30 μm of monascin groups are soft after 48h Osteocyte light absorption value is substantially less than 0nM groups, shows that 30 μm of monascin effect 48h have certain cartilage cell's toxicity.Pass through This experiment, it is determined that monascin is to the safe concentration of cartilage cell, i.e. 0~15 μm (Fig. 2).

(3) it is tested by Western blot, qPCR and ELISA, monascin can reduce soft under IL-1 β stimulations The content of the osteocyte cellular inflammation factor, it was confirmed that monascin has anti-inflammatory effect (Fig. 3) really.

(4) by Western blot, immunofluorescent test, show that monascin can reduce the inflammatory of extracellular matrix It degrades (Fig. 4).

(5) pass through further protein blot analysis of experiments, it has been found that monascin can be by inhibiting NF- κ B signals logical Anti-inflammatory effect (Fig. 5) is played in the activation on road, this result is also able to verification (Fig. 5) in the experiment of the cellular immunofluorescence of p65.

In addition, monascin of the present embodiment as known structure, it can be directly from market purchasing or chemical synthesis, containing red The pharmaceutical composition of aspergillin can be some long-acting or local sustained release preparations, for example, some of modern medicine can be utilized Biomaterial, including polycaprolactone, polylactic acid-polyglycol, polyvinyl alcohol etc. contain monascin, form compound system Agent.

SEQUENCE LISTING

<110>Attached second hospital of Wenzhou Medical University, attached Yu Ying children's hospitals of Wenzhou Medical University

<120>A kind of application of monascin in preparing treatment and delaying osteoarthritis drugs

<130> 2018

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213>It is artificial synthesized

<400> 1

tcctcacatc cctgagaacc 20

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<213>It is artificial synthesized

<400> 2

gtcgcacact ctgttgtgct 20

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<213>It is artificial synthesized

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gacgagacgg ataggcagag 20

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<213>It is artificial synthesized

<400> 4

cacatgcaag gaagggaact 20

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ccggagagga gacttcacag 20

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tccacgattt cccagagaac 20

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acggcatgga tctcaaagac 20

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gtgggtgagg agcacgtagt 20

Claims (5)

1. a kind of application of monascin in preparing treatment and delaying osteoarthritis drugs, the monascin molecular structural formula For shown in formula (I)：

2. application as described in claim 1, which is characterized in that the drug further includes pharmaceutical carrier, the pharmaceutical carrier choosing From micro emulsion and nano liposomes.

3. application as described in claim 1, which is characterized in that the drug is compound formulation, including erythromycin and is selected from Polycaprolactone, polylactic acid-polyglycol or polyvinyl alcohol contain material.

4. application as described in claim 1, which is characterized in that the dosage form of the drug is tablet, emulsion or capsule.

5. application according to any one of claims 1-4, which is characterized in that the erythromycin dosage of the drug is 5-10mg/ Kg weight.