CN109908138A - Application of the GL-V9 in preparation prevention and/or treatment colitis drug - Google Patents
Application of the GL-V9 in preparation prevention and/or treatment colitis drug Download PDFInfo
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- CN109908138A CN109908138A CN201910266100.1A CN201910266100A CN109908138A CN 109908138 A CN109908138 A CN 109908138A CN 201910266100 A CN201910266100 A CN 201910266100A CN 109908138 A CN109908138 A CN 109908138A
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Abstract
The invention discloses application of the GL-V9 in preparation prevention and/or treatment colitis drug.Compared with the existing technology, present invention finds in vitro experiment, GL-V9 can significantly inhibit secretion and the transcriptional level of the source of mouse macrophage RAW264.7 cellular inflammation factor of LPS induction.In vivo experiment, GL-V9 can improve the reduction of the colitis mice weight of DSS induction and the shortening of colon lengths, inhibit colonic tissue inflammatory cell infiltration and pathology damage.These effects show that GL-V9 can be used for the treatment of colitis, have the prospect of exploitation drug.
Description
Technical field
The invention discloses application of the GL-V9 in preparation prevention and/or treatment colitis drug, belong to GL-V9 and newly use
Way technical field.
Background content
Inflammation is that one kind that biological tissue is occurred by the stimulation of wound, the infection equivalent damage factor is with defense reaction
Main basic pathology process.All the time, inflammation is considered as a kind of mechanism of body self-protection.But inflammatory reaction continues
In the presence of will lead to body tissue damage, body normal physiological function is damaged.Inflammatory bowel disease (Inflammatory
Boweldisease, IBD) it is a kind of to show as the disorderly chronic nonspecific inflammatory bowel disease of intestinal inflammatory, including ulcer
Property colitis (Ulcer colitis, UC) and Crohn disease (Crohn ' s disease, CD).UC is a kind of morbidity in colon,
Patient is caused the intestines problem of the accomplished continuously or intermittently symptoms such as property diarrhea and hematochezia occur, lasting inflammation leads to the physiology function of colon
It can be badly damaged and cause dysfunction.And CD is a kind of systemic inflammatory diseases, site of pathological change spreads entire gastrointestinal tract, including food
There is discontinuous transmural inflammation in pipe, stomach, small intestine and colon, the entire intestinal wall of feature.The pathogenesis of research IBD is still unknown at present
Really, the factors such as disorder, gut barrier impaired, environment and tumor susceptibility gene may be adjusted with intestinal bacilli illness, Mucosal Immunity
It is related.Pathological experimental result and clinical evidence show that IBD is abnormal caused by the continuous action of chronic gut inflammation exempts from
Epidemic disease is reacted during inflammatory reaction continuous action, and panimmunity cell is participated, and body's immunity disorder is caused.It is huge
Phagocyte is as member important in immunocyte, and the overactivity of macrophage is to cause intestinal inflammatory anti-in inflammatory process
The main reason for answering.Therefore, for macrophage activation, safe and effective anti-inflammatory drug is found for treatment colitis and knot
Enteritis correlation colon cancer is of great significance.
Summary of the invention
Goal of the invention: in order to solve the above technical problems, the present invention provides GL-V9 in preparation prevention and/or treatment colon
Application in scorching drug, the drug for exploitation treatment colitis provide foundation.
Technical solution: to achieve the above object of the invention, technical solution of the present invention is as follows:
Application of the GL-V9 in preparation prevention and/or treatment colitis drug.
The GL-V9, molecular formula C24H27NO5, chemical structural formula is as follows:
The GL-V9 can significantly inhibit the secretion of the macrophage RAW264.7 cellular inflammation factor of LPS induction and turn
Record is horizontal.
The GL-V9 can improve the colitis of DSS induction.
Application of the composition containing GL-V9 in preparation prevention and/or treatment colitis drug.
The composition is using GL-V9 as active constituent, in addition drug made by pharmaceutically acceptable auxiliary material.
GL-V9 of the present invention can be used alone when treating colitis, and can also cooperate with other drugs makes simultaneously
With, or compound preparation use is made together with other drugs, it can achieve the purpose that treat colitis.
Pharmaceutically acceptable auxiliary material of the present invention refers to that the various routines needed for being added when preparing different dosage forms are auxiliary
Material, such as diluent, binder, disintegrating agent, glidant, lubricant, corrigent, inclusion material, adsorbent material etc. are with routine
Formulation method is prepared into any common oral preparation, for example, can be granule, powder, tablet, capsule, pill,
Oral solution, decoction, pill etc..
Present invention discover that GL-V9 has, the mouse weight for improving DSS induction mitigates, colon lengths shorten and colonic tissue disease
The function of degree of injury is managed, in vitro experiment, GL-V9 is able to suppress the source of mouse macrophage RAW264.7 cell of LPS stimulation
Inflammatory factor secretion.Therefore, GL-V9 can be used for treating colitis.
Technical effect: compared with the existing technology, present invention finds in vitro experiment, GL-V9 can significantly inhibit LPS
The secretion of the source of mouse macrophage RAW264.7 cellular inflammation factor of induction and transcriptional level.In vivo experiment, GL-V9 can
Improve DSS induction colitis mice weight reduction and colon lengths shortening, inhibit colonic tissue inflammatory cell infiltration and
Pathology damage.These effects show that different GL-V9 can be used for the treatment of colitis, have the prospect of exploitation drug.
Detailed description of the invention
Influence of Fig. 1 .GL-V9 to the LPS 264.7 cellular inflammation cytokine secretion of RAW induced, numerical value mean value ± standard
Difference indicates that there were significant differences compared to the blank group for P < 0.01 * *；There were significant differences compared with LPS model group for ##P < 0.01,
(Student's two-tailed t-test)；
Influence of Fig. 2 .GL-V9 to the LPS 264.7 cellular inflammation factor transcription level of RAW induced, numerical value mean value ±
Standard deviation indicates that there were significant differences compared to the blank group for P < 0.01 * *；There were significant differences compared with LPS model group for ##P < 0.01,
(Student's two-tailed t-test)；
Influence of Fig. 3 .GL-V9 to DSS model mice weight, numerical value indicates with means standard deviation, P < 0.01 * * and empty
White group compared to there were significant differences；There were significant differences compared with DSS model group for ##P < 0.01, (Student ' s two-tailedt-
test)；
Influence of Fig. 4 .GL-V9 to DSS inducing colitis mouse Colon length, numerical value indicates with means standard deviation, * * P
< 0.01 there were significant differences compared to the blank group；There were significant differences compared with DSS model group for ##P < 0.01, (Student ' stwo-
tailed t-test)；
Influence of Fig. 5 .GL-V9 to DSS model mice colonic tissue pathology damage；
Specific embodiment
With reference to the accompanying drawing and specific example, the present invention is furture elucidated.
Embodiment
One, test method
1.GL-V9 inhibits secretion and the transcriptional level of the source of mouse macrophage RAW264.7 cellular inflammation factor of LPS induction
1.1 experimental material
(1) drug
GL-V9(C24H27O5N, molecular weight: 409.47) being provided by China Medicine University, pale yellow powder, purity 98%,
Mother liquor, -20 DEG C of preservations are configured to DMSO.It is diluted to required concentration before use.
(2) cell strain
Source of mouse macrophage strain RAW 264.7 is purchased from Shanghai Life Sciences Research Institute, Chinese Academy Of Sciences' biochemistry and cell
Biological study institute cell bank, with containing 10% fetal calf serum DMEM culture solution culture.
(3) reagent
1) culture solution: DMEM culture medium is purchased from U.S. GIBCO company, and DMEM powder 10.4g is taken to be dissolved in 1000ml sterilizing three
It steams in water, 3.7g NaHCO is added3Adjust pH value to 7.2~7.4, cylindric style filter filtration sterilization, packing, 4 DEG C of refrigerators save.
Use preceding addition 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomysin.
2) fetal calf serum: U.S.'s GIBICO Products.Inactivate 30min through 56 DEG C of water-baths, dispense and be stored in -20 DEG C it is low
In temperature refrigerator.
3) PBS buffer solution: weighing NaCl 8.0g, KCl 0.20g, Na2HPO4H2O 1.56g, KH2PO4 2.0g, molten
In 1000mL tri-distilled water, high pressure sterilization, 4C refrigerator is saved.
4) DMSO solution: U.S. Sigma-Aldrich (St.Louis, Mo) Products.
5) LPS: it is purchased from Sigma Co., USA, is made into required concentration with cell culture fluid before experiment.
6) ELISA kit of mouse IL-1 β, IL-6 and TNF-α: it is purchased from Wuhan Boster Biological Technology Co., Ltd..
7) Real-time PCR related reagent: 0.1% pyrocarbonic acid diethyl ester (DEPC) is purchased from U.S. Amresco company, steams
Distilled water configures, and is put in after high pressure 3h spare in draught cupboard；RNA extracts Trizol kit and is purchased from Invitrogen company；cDNA
Synthetic agent box is purchased from TaKaRa company；Double-stranded DNA dyestuff SYBR Green PCR kit is purchased from TaKaRa company；Above and below
It swims primer and random primer is purchased from Shanghai Sheng Gong bioengineering Co., Ltd.
1.2 laboratory apparatus:
1) YJ-875 type Medical purification workbench: Suzhou Decontamination Equipment Plant's production.
2) 3111 type water-jacket typ CO2Incubator: U.S.'s Thermo electron Products.
3) electronic balance: Beijing Sai Duolisi instrument system Co., Ltd product.
4) LD4-2 generic centrifuge: Beijing Medical Centrifugal Machine Factory's product.
5) Centrifuge 5810R type centrifuge: German Eppendorf Products.
6) Varioskan all-wave length microplate reader is purchased from U.S. Thermo company；
7) 96 hole PCR amplification instrument of Mastercycler type is purchased from Eppendorf company, Germany.
8) Applied Biosystems 7500Fast Real-time PCR system is purchased from Perkin-Elmer company.
1.3 experimental methods:
(1) cells and supernatant ELISA is detected
After the GL-V9 of the LPS of 1 μ g/ml and various concentration are acted on 264.7 cell 12h of RAW, 4000rpm centrifugation is received
Collect cells and supernatant and carries out ELISA detection.Various samples are taken into 100 μ l, with mouse IL-1 β, the IL- of doctor's moral company
6 and TNF-α ELISA kit detection 264.7 cells and supernatant of RAW in IL-1 β, IL-6 and TNF-α content.Take 100
μ l standard items and sample are added in the plate hole of antibody embedding, and when every sub-sampling replaces pipette tips, are incubated for 1h at 37 DEG C, are removed each hole
Interior liquid is washed 3 times with the cleaning solution prepared, sequentially add biotinylated antibody, horseradish peroxidase label it is affine
Plain enzyme is eventually adding enzyme reaction substrate, developing solution and terminate liquid, detects each group absorbance (OD value) with wavelength 450nm.Streaming is thin
Born of the same parents' instrument detects apoptosis rate
(2) Real-time PCR experiment
Cell is after drug effect, vitellophag, cell is moved in the eppendorf pipe of removal RNase, 1000rpm
Cell is collected by centrifugation.After discarding PBS, 1ml RNAiso Plus is added and mixes, after being placed at room temperature for 5min, 200 μ l chloroforms are added,
30s is firmly shaked, is stored at room temperature 10min, subsequent 4 DEG C, 12000g is centrifuged 15min.Upper layer is shifted to new removal RNase's
In eppendorf pipe, isometric isopropanol is added, 10min is stored at room temperature after being mixed by inversion, subsequent 4 DEG C, 12000g is centrifuged
15min.Supernatant is abandoned, 75% ethanol washing of white RNA precipitate is vortexed and mixes, and 4 DEG C, 12000g is centrifuged 5min.Room temperature leeward
It is dry, the 20 processed water of μ l DEPC are added and dissolve RNA precipitate.Carry out reverse transcription reaction or being stored in after detection RNA concentration-
80 DEG C stand-by.
5 × gDNA Eraser Buffer, 2.0 μ l, 1.0 μ of gDNA Eraser is added by reverse transcription reagent box specification
l、Total RNA 1μl、RNase Free dH2O to 10 μ l of total volume.After 42 DEG C of incubation 2min, in 4 DEG C of coolings, 5 are added
×2 4.0 μ l of Buffer,1.0 μ l, RT Primer Mix of RT Enzyme Mix I ×
4 1.0 μ l and RNase Free dH2O to 20 μ l of total volume.37 DEG C of incubation 15min.85 DEG C of inactivation 15s terminate reaction.
PCR reaction system is added,10 μ l, PCR Forward Primer 0.8 of Premix Ex TaqTM II
0.8 0.4 μ l, cDNA solution of μ l, ROX Reference Dye II of μ l, PCR Reverse Primer 2 μ l, dH2O 6μl。
3 multiple holes of each measurement of concetration.
7500 type fluorescence quantitative PCR instrument of ABI sets PCR response procedures: initial denaturation, 50 DEG C of 2min；Unwinding, 95 DEG C
10min；PCR reaction, 95 DEG C of 15s, 60 circulations；60℃1min.Fluorescence real-time quantitative PCR data are collected, result is analyzed.
2.GL-V9 protection liver cell mitochondria function simultaneously inhibits liver cell internal oxidition stress
2.1 experimental material
(1) test medicine: GL-V9 is provided by Medicinal College, China Medical Univ., and purity is 99% or more, uses CMC before use
It is configured to required concentration gastric infusion.
(2) control drug: 5-aminosalicylic acid (5-ASA) is purchased from Sigma Co., USA, uses sodium carboxymethylcellulose
(CMC) it is configured to required concentration gastric infusion.
(3) experimental animal
Source, germline, strain: C57BL/6J mouse provides (experimental animal by this experimental animal Co., Ltd of Changzhou Cavan
Production licence: SCXK (Soviet Union) 2011-0003).
Week old: 6-8 weeks
Weight: 18-22g
Gender: female
2.2 experimental methods:
(1) experimental animal is grouped
(2) modeling method
The DSS solution for giving 3% mass volume ratio (w/v) (ensures that the lid of water bottle stoppers, and water outlet does not block, energy
Smoothness water outlet) freely drink 7 days after replace normal drinking water 3 days and establish acute colitis mouse model.Blank control group is always
Give normal drinking water nursing.It establishes from model the 1st day to the 10th day, the drug of corresponding dosage is given in medication therapy groups stomach-filling.
After eyeball of mouse excision in 11st day takes blood, cervical dislocation puts to death each group mouse, collects spleen and colon sample.
(3) DSS inducing mouse colitis model evaluation criterion
The observation of mouse ordinary circumstance, experiment start the ordinary circumstance of rear daily observation mouse, the state of mind, hair color variation,
Meal situation.Changes of weight observation, experiment start the weight and record that rear daily synchronization weighs each mouse.By every
Basal body mass when the every daily weight of mouse starts divided by modeling, obtains the percentage of same day changes of weight.Disease activity index is seen
It examines, daily observation of same time mouse stool form (whether stool shapes) and naked eyes bloody stool situation.
(4) DSS inducing colitis mouse Colon Histopathology assessment
After mouse Colon tissue is fixed with 10% formalin, paraffin embedding, conventional hematoxylin-eosin stains after dewaxing,
Optical microphotograph microscopic observation colonic tissue Pathologic changes.This experiment uses the paraffin section of tissue.Sample sections first exist
1h or so is placed in 60 DEG C of baking ovens, until the paraffin of slice is in drops.Then dewax in merging dimethylbenzene 10min, renews and matches
Dimethylbenzene, dewax 10min again.Sample sections are successively immersed in dehydrated alcohol 5min, 90% ethyl alcohol 2min, 70% ethyl alcohol
2min, distilled water 2min.Sample sections haematoxylin dyeing liquid is then dyed into 10min.PBS washes away extra dyeing liquor
10min, distilled water clean 10min again.95% ethyl alcohol 5s, eosin stains liquid dye 30s.Sample sections are then placed in 95% second
It is dehydrated 2min in alcohol, renews with 95% ethyl alcohol, is dehydrated 2min again.Sample sections are placed in dimethylbenzene again and impregnate 5min.It changes
Newly match dimethylbenzene, impregnates 5min, mountant mounting, microscopically observation again.
Two, test result
1, GL-V9 inhibits secretion and the transcriptional level of the source of mouse macrophage RAW264.7 cellular inflammation factor of LPS induction
After the GL-V9 of the LPS of 1 μ g/ml and various concentration are acted on 264.7 cell 12h of RAW, 4000rpm centrifugation is received
Collection cell and cells and supernatant are tested.Point of IL-1 β, IL-6 and TNF-α in ELISA experiment detection cells and supernatant
Secrete (Fig. 1).Real-time PCR experiment detects the transcriptional level (Fig. 2) of IL-1 β, IL-6 and TNF-α in cell.
Experimental result shows, such as Fig. 1 and Fig. 2,264.7 cell of RAW under LPS stimulation, IL-1 β, IL-6 and TNF-α
Secretion increases.After giving GL-V9, the secretion of above-mentioned inflammatory factor is significantly reduced.Real-time PCR is analysis shows LPS can be with
The transcription of 264.7 cellular inflammation factor IL-1 β of RAW, IL-6 and TNF-α is induced to increase, and GL-V9 significantly inhibits LPS induction
The transcription of these inflammatory factors.
2, GL-V9 improves the mouse colitis of DSS induction
The DSS solution for giving 3% mass volume ratio (w/v) (ensures that the lid of water bottle stoppers, and water outlet does not block, energy
Smoothness water outlet) freely drink 7 days after replace normal drinking water 3 days and establish acute colitis mouse model.Blank control group is always
Give normal drinking water nursing.It establishes from model the 1st day to the 10th day, the drug of corresponding dosage is given in medication therapy groups stomach-filling.
The changes of weight situation (Fig. 3) of record each group mouse daily.After eyeball of mouse excision in 11st day takes blood, cervical dislocation puts to death each group
Mouse collects spleen and colon sample.It measures mouse Colon length (Fig. 4).HE staining analysis mouse Colon histopathology changes
Become (Fig. 5).
GL-V9 is shown in Fig. 3 to the experimental treatment result of DSS inducing mouse colitis, 4,5.Experimental result is as follows, DSS mould
There is the symptom of obvious weight loss in type group mouse.After GL-V9 treatment, mouse weight starts gradually to go up.After testing,
Mouse Colon structure observation mouse Colon length is taken to change.DSS model group mouse is obviously shortened compared with blank group colon lengths, and
GL-V9 medication therapy groups mouse Colon length is restored to nearly normal level.Pathological analysis is carried out to experimental mice colonic tissue,
DSS model group mouse Colon mucous layer has hyperphlogosis cellular infiltration, and different degrees of necrosis occurs in part, and downright bad place has into
There is inflammatory cell infiltration in fibroblast proliferation, muscular layer of mucosa tissue；And GL-V9 group mouse Colon mucous membrane structure is restored just substantially
Often, epithelial structure is more complete, almost without inflammatory cell infiltration.5-ASA medication therapy groups mouse weight mitigates, colon lengths contract
Short and colonic tissue pathology damage also has a degree of improvement.
Claims (5)
- Application of the 1.GL-V9 in preparation prevention and/or treatment colitis drug.
- 2. application according to claim 1, which is characterized in that the macrophage that the GL-V9 can significantly inhibit LPS induction is thin The secretion of born of the same parents' RAW264.7 cellular inflammation factor and transcriptional level.
- 3. application according to claim 1, which is characterized in that the GL-V9 can improve the colitis of DSS induction.
- 4. the composition containing GL-V9 preparation prevention and/or or treatment colitis drug in application.
- 5. application according to claim 4, which is characterized in that the composition be using GL-V9 as active constituent, in addition Drug made by pharmaceutically acceptable auxiliary material.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113143909A (en) * | 2021-03-31 | 2021-07-23 | 中国药科大学 | Application of GL-V9 in preparation of medicine for preventing and/or treating hepatic fibrosis |
| CN113786402A (en) * | 2020-12-07 | 2021-12-14 | 中国药科大学 | Application of GL-V9 in preparation of medicine for preventing and/or treating sepsis |
| CN113786403A (en) * | 2020-12-07 | 2021-12-14 | 中国药科大学 | Application of GL-V9 in preparation of pancreatic cancer inhibition drug |
| CN115160279A (en) * | 2022-07-27 | 2022-10-11 | 中国药科大学 | Benzopyrone compound, pharmaceutical composition and application |
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2019
- 2019-04-03 CN CN201910266100.1A patent/CN109908138A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113786402A (en) * | 2020-12-07 | 2021-12-14 | 中国药科大学 | Application of GL-V9 in preparation of medicine for preventing and/or treating sepsis |
| CN113786403A (en) * | 2020-12-07 | 2021-12-14 | 中国药科大学 | Application of GL-V9 in preparation of pancreatic cancer inhibition drug |
| CN113143909A (en) * | 2021-03-31 | 2021-07-23 | 中国药科大学 | Application of GL-V9 in preparation of medicine for preventing and/or treating hepatic fibrosis |
| CN115160279A (en) * | 2022-07-27 | 2022-10-11 | 中国药科大学 | Benzopyrone compound, pharmaceutical composition and application |
| CN115160279B (en) * | 2022-07-27 | 2023-11-24 | 中国药科大学 | Benzopyrone compounds, pharmaceutical compositions and uses |
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Application publication date: 20190621 |