Background technique
Piperlongumine (Piperlongumine) is to be present in one of Piperaceae plant fructus piperis longi natural biological basesObject is closed, there is anti-malignant tumor, anti-platelet aggregation, adjusting blood lipid metabolism, anti-Parkinson's disease, promotion melanocyte to be proliferated,The effects of anti-inflammatory, antimycotic, and to normal cell, tissue, organ without apparent toxic side effect (referring to Li Lianjian, Chen ChunleiPiperlongumine pharmacotoxicological effect mechanism and the Hainan progress [J] medicine, 2015 (14): 2113-2115.).
It is horizontal that piperlongumine can increase intracellular reactive oxygen species (ROS), and selectively kills cancerCell., by inhibiting TrxR1 to inhibit gastric cancer, a kind of novel CRM1 inhibitor can press down in mankind mastopathy cell for itPI3K/Akt/mTOR processed can induce the cell death of pancreatic cancer cell (referring to Iwashita, Masaya, et al. "Piperlongumine,a constituent of Piper longum L.,inhibits rabbit plateletaggregation as a thromboxane A2receptor antagonist."European journal ofpharmacology 570.1-3(2007):38-42.).Piperlongumine, can be with as a kind of thromboxane A (2) receptor antagonistInhibit platelet aggregation (referring to Iwashita, Masaya, et al. " Piperlongumine, a constituent ofPiper longum L.,inhibits rabbit platelet aggregation as a thromboxaneA2receptor antagonist."European journal of pharmacology 570.1-3(2007):38-42.).Piperlongumine also can be by inhibiting Akt/mTOR signal path to promote cell autophagy, and the dead (ginseng of mediate tumor cellSee Ryu, Jahee, et al. " Piperlongumine as a potential activator of AMP-activatedprotein kinase in HepG2cells."Natural product research 28.22(2014):2040-2043.).Piperlongumine causes the growth inhibition of interior tumor cell, without changing biochemistry, hematology and histopathologyParameter.(referring to Bezerra, Daniel P., et al. " In vivo growth inhibition of sarcoma 180bypiperlonguminine,an alkaloid amide from the Piper species."Journal of Appliedtoxicology 28.5(2008):599-607.).Therefore, the medical usage of piperlongumine can be developed further.
Patent document piperlongumine analog, preparation method and applications (CN104910174A) disclose piperlongumine classThe drug for treating or preventing thrombotic disease is used to prepare like object；Patent document piperlongumine is preparing medicine for treating tumor metastasisUtilization (CN104415035A) in object discloses piperlongumine and its pharmaceutical salts and is preparing the utilization in medicine for anti transfer of tumor.
It will lead to cell after bacteria-infected cells and generate inflammatory reaction, further result in Apoptosis, seriously affect the mankindHealth.Therefore, researching and developing a variety of antibacterial-anti-inflammatory drugs is the key method for solving bacterium infection.Fusobacterium nucleatum(Fusobacterium nucleatum, Fn) belongs to Gram-negative without gemma fusiform bacilarmature, and obligate anaerobe is periodontitisOne of main pathogenic bacteria.Fusobacterium nucleatum is a kind of with periodontosis and body other each site disorders have the cause of disease of close tiesBacterium.
Currently, piperlongumine is mainly used for the medicines such as antitumor, treatment or prevention thrombotic disease according to the prior artThe preparation of object, there is not yet in relation to piperlongumine in the report for preparing the application in antibacterial-anti-inflammatory drug.
Summary of the invention
The object of the present invention is to provide the new opplications of piperlongumine, and specially piperlongumine is in preparing antibacterial-anti-inflammatory drugApplication.
Term explanation:
The structural formula of piperlongumine of the present invention is as follows, and market is commercially available or presses prior art chemical synthesis system?.
Technical scheme is as follows:
Piperlongumine is preparing the application in antibacterial-anti-inflammatory drug.
Preferred according to the present invention, in the application, the antibacterial refers to that Fusobacterium nucleatum Fn, i.e. piperlongumine are makingApplication in standby anti-Fusobacterium nucleatum Fn drug.
Above-mentioned piperlongumine includes piperlongumine and piperlongumine clinically acceptable salt, contains piperlongumineCompound medicament composition and clinically acceptable preparation.The preparation formulation can be injection, solution, emulsion, mouthTake liquid, suspension, paste, creme, spray, drops, freeze drying powder injection, powder, granule, pill, tablet, patch etc..
In application of the present invention, piperlongumine be used alone or with other antibacterial-anti-inflammatory drug use in conjunction.It is excellentChoosing, the antibacterial-anti-inflammatory drug includes metronidazole, gentamicin, azithromycin, Imipenem, Meropenem, promise fluorine sandOne or more of star.
Beneficial effect
The present invention is based on the transcript profile data that Fusobacterium nucleatum Fn bacterium infection HGF cell generates, and are relocated based on drugAnalysis software cogena finds drug candidate piperlongumine, related to a large amount of inflammation logical in conjunction with differential gene in path analysis resultRoad is highly relevant, it was initially believed that piperlongumine has antibacterial and anti-inflammation functions.It only used Fusobacterium nucleatum Fn in the present invention, stillThe drug can be generalized in other bacteriums.Therefore, the present invention extends the pharmaceutical applications of piperlongumine, discloses piperlongumineThe application in antibacterial-anti-inflammatory drug is being prepared, is providing a kind of new therapeutic choice for clinical treatment inflammation disease.
Specific embodiment
Following embodiments are for further illustrating but being not limited to the present invention.
1. materials and methods:
1.1 bacteriums and cell
Fusobacterium nucleatum (F.nucleatum, Fn, ATCC 25586) is frozen from oral tissue regeneration key lab, Shandong ProvinceDeposit strain library acquisition.
Gingival Fibroblasts (HGF): isolated from 6 18-30 years old extraction of impacted third molar volunteer patients.
1.2 reagent
It defibrinates sheep blood (Hai Bo biotech firm, Qingdao, Shandong, China), PBS (Suo Laibao, Beijing, China), chlorineChange ferroheme-vitamin K1 (Hai Bo biotech firm, Qingdao, Shandong, China), brain heart infusion blood meida (Suo Laibao, Beijing,China), BHI fluid nutrient medium (Suo Laibao, Beijing, China), collagenase type I (Suo Laibao, Beijing, China), DispaseII pointsDissipate enzyme (Invitrogen, Carlsbad, CA, USA), reactive oxygen species detection kit (Bei Bo, Shanghai), piperlongumine(MedChemExpress, Shanghai).
1.3 instrument
(Austria contains public for anaerobic culture box (Britain's DWS DG250- compact anaerobism work station, Britain), ultraviolet specrophotometerDepartment, Hangzhou, Zhejiang, China), qPCR instrument (Roche, Basel, Switzerland), T25 culture bottle (Corning company, beautyState).
1.4 experimental design
People's Gingival Fibroblasts cell is infected by building Fusobacterium nucleatum Fn, bacteria-infected cells model is established, pressesAccording to 0,
2,6,12,24,48 hours acquisition cells extract RNA and carry out the transcriptome analysis based on two generation sequence rna-Seq,The gene expression spectrum signature of bacteria-infected cells is obtained, based on coexpression KEGG access and the joint enrichment point of CMap drug gene collectionAnalysis, is found to have the drug candidate list of antibacterial and anti-inflammation functions.
1.5 experimentation
1.5.1 Fusobacterium nucleatum Fn separation, culture, identification
Defrosting Fusobacterium nucleatum (F.nucleatum, ATCC 25586) strain is inoculated in and defibrinates sheep containing 10%Blood, 0.5% hemin-vitamin K1 brain heart infusion blood meida on, be placed in 37 DEG C of anaerobic culture boxes be incubated for 48h extremelyGrow bacterium colony, picking single colonie is placed in 100mL BHI fluid nutrient medium proliferation to logarithmic growth phase, 6000rpm × 5min fromThe fresh bacterium solution of the heart, sterile PBS washing thalline 2 times are resuspended in BHI broth, and ultraviolet specrophotometer measures OD600nm absorbanceValue is completed to convert between OD value and number of bacteria, be identified using special primer row qPCR, it is standby to obtain Fusobacterium nucleatum (see Fig. 1)With.The DNA of bacteria is extracted, by PCR amplification, amplified production send Hua Da gene Co., Ltd to carry out 16S sequencing, sequencing resultIt is compared in HOMD database, is identified as 25586 bacterial strain of FN.
1.5.2 people's Gingival Fibroblasts HGF is separately cultured
Recruit 18-30 year old extraction of impacted third molar volunteer patient 6, informed consent, acquisition gingiva tissue.In vitro gum groupIt knits and is dipped in sterile PBS, rapidly from clinical metastasis to laboratory, rinsed in sterile super-clean bench expert PBS, it is big to shred into 1-3mm2Fractionlet collects fragment in sterile EP tube, and collagenase type I and DispaseII dispase digestion 2h hang postdigestive cellLiquid is placed in T25 culture bottle, and 37 DEG C, 5%CO2Incubator is incubated for about 7-10 days, and cell growth, proliferation is reached to cell confluency80%-90% is passed on 1:3 dilution ratio, expands culture.Cell grew into for the 4th generation, saved, spare (see Fig. 2).
1.5.3. Fusobacterium nucleatum Fn handles people's Gingival Fibroblasts HGF, carries out transcript profile sequencing analysis
The P4 of 5 patients is cultivated simultaneously for HGF, and pancreatin digests, and blood counting chamber counts, and is inoculated in 6 orifice plates respectively(2X105The hole cell/), after cell adherent growth, HGF (Fn:HGF=100:1) is infected with Fn, is uninfected by a group conduct control,Infection time is respectively 2,6,12,24,48 hours, after infection, and with Trizol lytic cell, cell pyrolysis liquid is received respectivelyCombine in no enzyme EP pipe, number (cell origin number is B, C, D, E, F, control group C, experimental group F, the time with it is digital comeIt indicates, such as: B2C represents cell derived from B patient, and 2 represent 2h, and C represents untreated fish group), send Hua Da gene Co., Ltd to carry outSequencing based on RNA-Seq obtains the transcript profile feature of the time series of bacteria-infected cells.
1.5.4 based on coexpression access and drug gene collection Conjoint Analysis discovery bacteria-infected cells related pathways andDrug candidate
The co-expression gene collection enrichment analysis R software package cogena that coexpression enrichment analysis is developed before using applicant,Select K-means clustering method, clustering cluster 3.Pathway gene collection selects KEGG signal path, and drug gene collection selects CMap medicineObject collection, statistical test method are hypergeometric distribution hypothesis testing method.It is presented and is tied based on thermal map, access or drug-rich figureFruit.
2. experimental result
The changes in gene expression feature of 2.1Fn bacterium infection people's Gingival Fibroblasts HGF
For different time points (2,6,12,24,48 hours), it is based respectively on limma software package and analyzes to obtain differential expressionGene takes the intersection of differential gene to obtain 971 genes and thermal map (see Fig. 3) is presented using cogena, can more see on a macro scaleChanges in gene expression feature after to bacteria-infected cells, horizontal axis is the sample changed according to natural time, longitudinal axis generation in the figureTable gene expression profile is broadly divided into three coexpression clustering clusters (cluster), and wherein clustering cluster 1 and 2 is after bacteria-infected cellsUp-regulation gene cluster, 3 are down-regulated gene cluster.
2.2 coexpression path analysis are several immune and are metabolized relevant access
It is analyzed based on above-mentioned thermal map as a result, carrying out co-expression gene KEGG signal path analysis (Fig. 4), discovery is largely exempted fromEpidemic disease, bacterium infection and the relevant signal path of metabolism, such as cell factor and cytokine receptor interaction access (cytokineCytokine-receptor interaction pathway), Helicobacter pylori infection epithelial cell signal path(epithelial cell signaling in helicobacter pylori infection pathway), Li Shiman are formerInsect infection signal path (leishmania infection pathway), glutathione metabolism access (glutathionMetabolism pathway) etc..
2.3 coexpression drug reorientation analyses
By thermal map and access enrichment figure, finds the gene expression spectrum signature of Fn bacterium infection HGF cell, use cogenaSoftware carries out calculating drug reorientation analysis, (other candidate medicines of part as a result as shown in Figure 5 for co-expression gene clustering cluster 2Object has blocked), demonstrate drug Rimexolone (Rimexolone, the 5th) and the antibacterial agent chlorine for treating Inflammatory eye conditionsChange the antibacterial action of benzyl ethoxy ammonium (methylbenzethonium chloride, the 7th), finds piperlongumine(Piperlongumine) enrichment analysis result the 8th is ranked.It is enriched in conjunction with a large amount of inflammatory reactions in coexpression path analysisAs a result, piperlongumine has antibacterial and anti-inflammation functions, it is expected to develop into the drug with antibacterial and anti-inflammation functions.Therefore, piperlongumineIt can be applied to prepare in antibacterial-anti-inflammatory drug.