CN110028574A - Application of the rotenone in activation channel catfish nuclear receptor PXR and cytochromes enzyme CYP3A activity - Google Patents
Application of the rotenone in activation channel catfish nuclear receptor PXR and cytochromes enzyme CYP3A activity Download PDFInfo
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- CN110028574A CN110028574A CN201910274735.6A CN201910274735A CN110028574A CN 110028574 A CN110028574 A CN 110028574A CN 201910274735 A CN201910274735 A CN 201910274735A CN 110028574 A CN110028574 A CN 110028574A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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Abstract
The present invention has studied channel catfish and respectively organizes center receptor PXR and cytochromes enzyme CYP3A expression, filtering out for detecting the active destination organization of PXR and CYP3A is respectively the gill and anterior intestine, the gill and anterior intestine are acquired after filling channel catfish using the monomer mouth purified from natural product extraction, detect the expression of nuclear receptor PXR and Cytochrome P450 3A enzyme gene, filter out the natural products monomer rotenone that can regulate and control CYP3A enzyme by activation nuclear receptor PXR access, it is verified by experiments, rotenone can make residence time of the Enrofloxacin in channel catfish belt leather muscle (Edible tissues) shorten to 7 d by 12 d, the residual quantity at same time point is significantly reduced, therefore, rotenone can be used for accelerating quinolones chemicals in channel catfish body residual The elimination stayed, for ensureing fish quality, the risk for reducing aquatic products of the human body intake containing chemicals residue is of great significance.
Description
Technical field
The invention belongs to aquatic livestock pharmacological techniques fields, and in particular to rotenone is in activation channel catfish nuclear receptor
Application in PXR and cytochromes enzyme CYP3A activity.
Background technique
Channel catfish (Ietalurus punetaus) originates in North America, belongs to siluriformes (Silurfornes), Channel-catfish section
(Ictaluridae), in 1984 by Hubei introduce and at home cultivate on a large scale.Channel catfish is that a kind of large size is light
Water fish class has many advantages, such as that growth is fast, wide adaptability, premunition is strong, meat is superior and cheap, the deep happiness by consumer
Love.Cut-off 2015, since the channel catfish annual output of cultivation is higher than the U.S., China has become channel catfish cultivation first
Big country.But along with the expansion of cultivation scale, the situations such as raising, variety deterioration, drug abuse of intensive culture degree go out
Existing, the use demand of drug is also being continuously increased.Medicament residue problem, which has become, hinders the industry development of China's channel catfish
One of critical issue.
Many receptors have proved to be the key factor for mediating organism drug metabolic enzyme at present, such as nuclear receptor PXR
CYP3A can be induced under conditions of forming dimer with retinoid X receptors RXR.Drug metabolic enzyme and related core by
Body plays a key effect in drug metabolism and residual.PXR is the NR1I subfamily member of nuclear receptor superfamily (NR), 1998
The full-length cDNA of PXR albumen clones from mouse obtain for the first time, and Given this receptor can be by a series of native compounds or synthesis
Pregnane activated, therefore be named as Pregnane X Receptor.PXR be by ligand binding domain (ligand binding domain,
LBD it) is formed with DNA binding domain (dna binding domain, DBD).Different from other nuclear receptors, the LBD structure of PXR is more
Special, spheroidal binding cavity, this chamber has potential scalability, therefore can accommodate the ligand of all size, has potential
Scalability, it is related with the popularity of PXR ligand.Since LBD sequence has compared with low homology, PXR regulation is special in different germlines
Property also has large change.
All biologies all have metabolizing enzymes, by fat-soluble organic substance be converted to it is water-soluble, be easily drained
Metabolite.Metabolic enzyme is almost present in the intracorporal various tissues of biology, for aquatile, content highest in liver,
CYP3A is the main enzyme for participating in drug metabolism.The CYP3A regulatory pathway that PXR is mediated is considered as clinical medicine interaction
Important molecule basis.The research of CYP3A focuses mostly in mammal, also very poor to the research of fish.The present invention is to spot
The expression of nuclear receptor PXR and CYP3A are analyzed with distribution in point fork tail Channel-catfish tissue, and activate nuclear receptor PXR access to having
And then the natural products ligand for raising CYP3A gene expression function is screened, it is intended to for drug in regulation channel catfish body
Residual provides technical support, and then ensures that the quality safety of aquatic products is of great significance.
Summary of the invention
The object of the present invention is to provide rotenone in activation channel catfish nuclear receptor PXR and cytochromes enzyme
Application in CYP3A activity, filters out with activation nuclear receptor PXR access and then raises the natural products of CYP3A gene expression
Ligand, and be applied to acceleration and eliminate quinolones medicament relict in channel catfish body.
In order to achieve the above object, the present invention takes following technical measures:
The expression and distribution feature of PXR and CYP3A gene in channel catfish tissue:
It has studied channel catfish and respectively organizes center receptor PXR and cytochromes enzyme CYP3A expression, nuclear receptor PXR
Gene expressed in channel catfish tissue the sequence that arranges from high to low be the gill, kidney, hindgut, heart, anterior intestine, spleen, middle intestines,
Brain, skin, liver, muscle, CYP3A gene expressed in channel catfish is respectively organized the sequence that arranges from high to low be anterior intestine, hindgut,
Middle intestines, the gill, liver, kidney, skin, spleen, brain, heart, muscle have filtered out living for detecting PXR and CYP3A in channel catfish tissue
Property destination organization be respectively the gill and anterior intestine, for subsequent development screen nuclear receptor activities activate natural products provide technical foundation and
Destination organization.
The screening of channel catfish nuclear receptor PXR activation ligand natural products monomer:
The gill and anterior intestine that acquisition channel catfish after channel catfish is filled using natural products monomer mouth, detect nuclear receptor
The expression of PXR and cromoci YP3A enzyme gene, filtering out can be by activation nuclear receptor PXR access regulation
The natural products monomer rotenone of CYP3A enzyme.
Channel catfish nuclear receptor PXR is activated to accelerate the application in channel catfish body in chemicals residue:
It is verified by experiments, rotenone can make residual of the Enrofloxacin in channel catfish belt leather muscle (Edible tissues)
Time shortens to 7d by 12d, and the residual quantity at same time point is significantly reduced, and therefore, rotenone can be used for accelerating channel catfish
The elimination of quinolones chemicals residue in Channel-catfish body, quinolone drugs include Enrofloxacin, Ofloxacin, Dan Nuosha star,
Oxolinic acid.
Compared with prior art, the present invention has the following advantages and beneficial effects:
(1) present invention has studied channel catfish for the first time and respectively organizes center receptor PXR and cytochromes enzyme CYP3A expression of enzymes
Level, finding out the higher tissue of nuclear receptor expression as screening by expression has activation nuclear receptor PXR and cell
" destination organization " of the natural products of pigment enzyme CYP3A enzyme effect.
(2) the natural production with activation channel catfish nuclear receptor PXR and cytochromes enzyme CYP3A enzyme that the present invention screens
Ligand of the object monomer as nuclear receptor PXR, the nuclear receptor PXR after activation regulate and control the activity of cytochromes enzyme CYP3A enzyme
(up-regulation) accelerates chemicals in the intracorporal residual eliminating of channel catfish.Such as trifoliate jewelvine one monomers can make Enrofloxacin exist
Residence time in channel catfish belt leather muscle (Edible tissues) shortens to 7d by 12d, and the residence time shortens original 5/
12, the residual quantity at same time point is significantly reduced.
(3) present invention can be such that the residence time of chemicals in aquatic products shortens reduces with residual quantity, for ensureing aquatic products
Quality safety, the risk for reducing aquatic products of the human body intake containing chemicals residue are of great significance.
Detailed description of the invention
Fig. 1 is the agarose gel electrophoresis figure of primer screening.The best primer PXR-3F/ of nuclear receptor PXR gene of A- screening
The best primer CYP3A-4F/4R of CYP3A gene of 3R, B- screening.
Fig. 2 is the amplification standard curve of primer.The amplification standard curve of A- primer PXR-3F/3R, B- primer CYP3A-4F/
The amplification standard curve of 4R.
Fig. 3 channel catfish respectively organize in PXR and CYP3A gene relative expression quantity.A-PXR gene is in channel catfish
Relative expression quantity in tissue, relative expression quantity of the B-CYP3A gene in channel catfish tissue.
Enrofloxacin residual depletion role (1-288h) in Fig. 4-channel catfish belt leather muscle.A- mouthfuls of filling Enrofloxacins exist
Elimination regularity in channel catfish belt leather muscle；B- mouthfuls fill the rotenone with activation nuclear receptor PXR of screening to spot
Enrofloxacin residual depletion role influences in Cha Wei Channel-catfish belt leather muscle.
Specific embodiment
Embodiment 1
The expression and distribution feature of PXR and CYP3A gene in channel catfish tissue:
According to Trizol (Invitrogen) specification extract channel catfish liver, kidney, skin, muscle, brain, the heart, the gill,
Spleen, front court, middle intestines, hindgut etc. organize RNA.Each total tissue RNA of the channel catfish of extraction is used into Dnase I respectively
(Takara) after handling, the concentration and quality (A λ 260/A λ 280) of proposed RNA are detected with spectrophotometer.It is tried using reverse transcription
Agent box Prime ScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time) is inverted
Record synthesis the first chain of cDNA.
The screening of primer: according to the cDNA of channel catfish PXR and the CYP3A gene of NCBI GenBank warehouse publication
Full length sequence devises specificity by Primer5.0, Primer quest tool and raw work bioengineering Photographing On-line
Amplification of the primer for regular-PCR and real-time quantitative PCR, primer sequence are shown in Table 1.Respectively by the positive primer of design, anti-primer,
2 × hieff, DEPC water and the mixing of cDNA template, 60 DEG C of reaction 1h, agarose electrophoresis detection.It is screening with the synthetic quantity of cDNA
Standard screen selects the primer of PXR and CYP3A, respectively PXR-3F/3R, CYP3A-4F/4R.Again by channel catfish cDNA template
It is diluted to five concentration gradients (extension rate is respectively as follows: initial concentration, 5,25,125,625 times), is carried out by primer glimmering in real time
Fluorescent Quantitative PCR obtains the standard curve of primer amplification, judges whether experimental condition reaches requirement of experiment.It is optimized, PXR and
The amplification efficiency of CYP3A gene is respectively 100.64% and 95.16%, and slope is respectively -3.307 and -3.444, and showing can be with
With 2Δ Δ CTMethod carries out relative quantitative assay.There is no miscellaneous peak appearance in solubility curve, does not occur non-specific amplification, experimental condition
Reach requirement of experiment, reliability is higher.
The expression of PXR and CYP3A gene: real-time quantitative expression analysis is carried out with quantitative PCR apparatus.PCR reaction condition according to
The method that fluorescence quantitative kit is recommended carries out, and 18S rRNA is as reference gene.PCR after reaction, is expanded and is melted
Solution curve analysis.It is control with the expression quantity of heart, carries out significance analysis with SPSS19.0 software, data are with average value ± mark
Quasi- difference indicates that P < 0.05 is significant difference.
1 primer of table and its sequence
As shown in figure 3, expression of the PXR in each tissue of channel catfish be successively the gill, kidney, hindgut, heart,
Anterior intestine, spleen, middle intestines, brain, skin, liver, muscle.PXR expression is higher in the gill, kidney, with hindgut, heart, anterior intestine, spleen, middle intestines,
Brain, skin, liver, there are significant difference (P < 0.05) between muscle；Also there were significant differences between the gill and kidney (P < 0.05)；It is again
In the heart, spleen, anterior intestine, hindgut, there was no significant difference (P > 0.05)；The heart, skin, liver, muscle, spleen, brain, anterior intestine, between middle intestines without aobvious
It writes sex differernce (P > 0.05).Expression of the CYP3A in each tissue of channel catfish be successively anterior intestine, hindgut, middle intestines,
The gill, liver,kidney,spleen, skin, brain, the heart, muscle；Wherein CYP3A is higher in anterior intestine, middle intestines, the expression of hindgut, with its hetero-organization
There are significant difference (P < 0.05)；There were significant differences between anterior intestine and middle intestines, hindgut (P < 0.05), and middle intestines and hindgut are without significant
Difference (P < 0.05)；The gill and kidney again, spleen, skin, brain, the heart, there were significant differences (P < 0.05) between muscle, but the gill and liver it
Between there was no significant difference (P > 0.05)；Liver,kidney,spleen, skin, brain, the heart, there was no significant difference (P > 0.05) between muscle.Pass through
Experiment and interpretation of result have been filtered out is respectively for detecting the active destination organization of PXR and CYP3A in channel catfish tissue
The gill and anterior intestine.
Embodiment 2
Channel catfish mouth fills natural products monomer aureusidin, catechin, delphinidin, Cyanidin, ginkgetin, fish
Rattan ketone, daidzein, Puerarin, silymarin, hesperetin, Quercetin, appointing in baicalein (can pass through commercially available acquisition)
After one kind of anticipating, the gill and foregut tissues of channel catfish are acquired, detects the expression of channel catfish nuclear receptor PXR and CYP3A, sieve
Select the natural products with activation nuclear receptor PXR effect as ligand, the results showed that, rotenone can be used as activation nuclear receptor PXR
Natural products ligand, and the receptor PXR activated can be with the activity of regulating cell pigment enzyme CYP3A enzyme.
Natural products rotenone by testing further verifying screening is accelerating to eliminate chemical drugs in channel catfish body
Effect in object residual, channel catfish is randomly divided into experimental group and control group: experimental group channel catfish is first by 10mg/kg fish
After weight single mouth fills Enrofloxacin 4h, after the natural products rotenone that screening is filled by 40mg/kg fish body weight mouth, grace promise is filled with mouth
The time of Sha Xing be starting point respectively at 1h, 2h, 4h, 6h, 8h, 12h, for 24 hours, 48h, 72h, 96h, 120h, 144h, 168h, 216h,
288h acquires experimental group channel catfish belt leather muscle；Control group channel catfish fills En Nuosha by 10mg/kg weight single mouth
1h, 2h after star, 4h, 6h, 8h, 12h, for 24 hours, 48h, 72h, 96h, 120h, 144h, 168h, 216h, 288h acquire control group spot
Cha Wei Channel-catfish belt leather muscle.It is to be measured by homogeneous rear -20 DEG C of preservations of the channel catfish belt leather muscle homogenizer of acquisition.In sample
The determination of residual amount of Enrofloxacin is referring to No. 1077 bulletin -1-2008 " aquatic products of National Standard of the People's Republic of China-Ministry of Agriculture
In 17 kinds of sulfamidos and the measurement Liquid Chromatography-Tandem Mass Spectrometry of 15 kinds of quinolones medicament relict amounts " carry out.Comparative experiments group
With enrofloxacin residual amount and residual eliminating time in control group channel catfish belt leather muscle, experimental group spot is pitched as the result is shown
Enrofloxacin residual amount is residual lower than Enrofloxacin in control group and experimental group channel catfish belt leather muscle in Wei Channel-catfish belt leather muscle
The elimination time is stayed to be faster than the residence time in control group channel catfish belt leather muscle.
The effect mainly by drug metabolic enzyme is eliminated in the metabolism of drug in vivo, and the activity of drug metabolic enzyme is main
It is to be regulated and controled by nuclear receptor.After the natural products rotenone activation nuclear receptor PXR filtered out in this experiment, and nuclear receptor PXR is logical
It crosses the activity of regulation (up-regulation) drug metabolic enzyme CYP3A enzyme and then promotes the metabolism and elimination of drug.Due to quinolone drugs
The structural formula of Ofloxacin, Dan Nuosha star, oxolinic acid etc. is similar to Enrofloxacin, such medicine of drug metabolic enzyme CYP3A enzymatic
The key reaction that object occurs is similar, and the activity of drug metabolic enzyme is higher, and the rate that these drugs are metabolized is faster, and drug is in fish body
The interior remaining time is also shorter.
Claims (3)
1. application of the rotenone in activation channel catfish nuclear receptor PXR and cytochromes enzyme CYP3A activity.
2. rotenone described in claim 1 is accelerating to eliminate the application in channel catfish body in quinolones medicament relict.
3. rotenone according to claim 2 is accelerating to eliminate answering in quinolones medicament relict in channel catfish body
With, which is characterized in that quinolone drugs includes Enrofloxacin, Ofloxacin, Dan Nuosha star, oxolinic acid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111521694A (en) * | 2020-04-10 | 2020-08-11 | 中国水产科学研究院长江水产研究所 | Application of ligustilide in eliminating quinolone drug residues in cultured fishes |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101647524A (en) * | 2009-08-14 | 2010-02-17 | 淮安华威饲料发展有限公司 | Feed for controlling medicine residues of channel catfishes |
| CN104267125A (en) * | 2014-10-13 | 2015-01-07 | 中国水产科学研究院长江水产研究所 | Method for predicting drug residues in grass carp tissue with physiological pharmacokinetic model |
Non-Patent Citations (4)
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| HU D等: "Artemisinin protects against dextran sulfate-sodium-induced inflammatory bowel disease, which is associated with activation of the pregnane X receptor", 《EUROPEAN JOURNAL OF PHARMACOLOGY》, vol. 738, 5 September 2014 (2014-09-05), pages 273 - 284 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111521694A (en) * | 2020-04-10 | 2020-08-11 | 中国水产科学研究院长江水产研究所 | Application of ligustilide in eliminating quinolone drug residues in cultured fishes |
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Application publication date: 20190719 |