CN104142384A - Method for screening active compounds capable of protecting or improving renal functions - Google Patents
Method for screening active compounds capable of protecting or improving renal functions Download PDFInfo
- Publication number
- CN104142384A CN104142384A CN201410377736.0A CN201410377736A CN104142384A CN 104142384 A CN104142384 A CN 104142384A CN 201410377736 A CN201410377736 A CN 201410377736A CN 104142384 A CN104142384 A CN 104142384A
- Authority
- CN
- China
- Prior art keywords
- embryo
- group
- compound
- water
- creatinine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method for screening active compounds capable of protecting or improving renal functions. According to the method, compounds which can cause kidney injuries and compounds to be tested together act on zebra fish embryos, or compounds with renal toxicity act on the zebra fish embryos first to cause kidney injuries and then the compounds to be tested are used for processing the injured embryos, changes of creatinine content in embryo tissue are used as indexes for detecting renal functions, and whether the compounds to be tested have activity to protect or improve the renal functions is analyzed. The active compound screening method which is quick, reliable and high in pass is established, and lead compounds are provided for research and development of treatment drugs of kidney diseases.
Description
Technical field
The present invention relates to a kind of method of screening protection or improving renal function reactive compound, belong to medical biotechnology field.
Research background
Along with social development, rhythm of life is accelerated, add eating habit and life style that people are bad, cause the chronic disease incidences of disease such as diabetes, hypertension, heart disease obviously to increase, these diseases need to be taken medicine conventionally throughout the year, easily renal function are caused to damage, simultaneously, the day by day serious environmental pollution that rapid economic development brings, has further aggravated the generation of kidney injury.Clinically, renal lesions has onset concealment, treats the feature difficult, mortality ratio is high, useful clinically methods for the treatment of and medicine are very limited, be developed to later stage patient and can only rely on the even kidney transplantation of dialysing to sustain life, bring huge misery and heavy financial burden to patient and family thereof.Therefore, establish screening technique fast and effectively, accelerate the research and development of original new drug, prevention and treatment to kidney trouble are significant.
In the body that utilizes whole animal model to carry out, test, given the test agent is placed under complicated organismic internal environment, can objectively responds sample metabolic rule and mechanism of action in vivo, and the reaction of the interior each organ interaction lower body of body, the result reliability obtaining is high, and reference value is large.But these animal pattern feeding costs are high, the breeding cycle is long, be difficult to realize quick, the high flux screening of compound activity.Zebra fish, as a kind of desirable vertebra model organism, is used widely in fields such as Developmental Biology, disease model and medicament research and development.With other mammal model ratios, zebra fish egg laying amount is high, and growth cycle is short, and drug dose is few, repeatable strong, is more suitable for the screening study of large-scale compound.
Kidney has three kinds of forms in evolution: pronephridiostome, middle kidney and metanephros, at zebra fish species, kidney is divided into pronephridiostome and the middle kidney of adult fish phase of young stage.14h after embryo fertilization (14hpf, 14hours post-fertilization) zebra fish kidney germinates, and when 72hpf, kidney has been grown.Zebra fish pronephridiostome is made up of a pair of nephron, and the anatomical structure of pronephridiostome is simpler than the middle kidney of adult fish and mammiferous metanephros, but similar to mammiferous kidney on cell composition and molecular level, and has possessed the biological function of same complexity.And the zebrafish embryo kidney reaction that chemical compound lot causes reacts closely similar with the mankind's kidney.In addition, zebra fish grew in first week, because the gill is not also grown completely, the main kidney that relies on is discharged unnecessary moisture and metabolic waste in body, in addition prelarva needn't can survive by feeding this in stage, and disturbing factor is few, is very beneficial for carrying out the research of kidney related fields.
Disease causes body pathology physiology to change, and must cause the variation of metabolic components content in tissue, to these components, more especially with the detection of the closely-related biomarker of organ dysfunction state, is the important evidence that diagnoses the illness generations, develops.Creatinine is the metabolic product of muscle, is transformed by precursor compound phosphocreatine, is mainly excreted by kidney.When impaired renal function, creatinine is accumulated in vivo becomes harmful toxins.Clinically, serum creatinine value is to understand the important indicator of renal function, in the kidney related experiment of carrying out with rodent models, and blood, the UCr value test item that is absolutely necessary.To zebrafish embryo, because volume is little, be difficult to obtain enough blood or urine specimen, at present, there is no report for the detection data of zebrafish embryo creatinine aspect, but there are some researches show, in one section of developmental stage after embryo fertilization, creatinine content in embryonic tissue is progressively ascendant trend, and this strengthens relevantly with embryo motion ability gradually, and also the functional status of the creatinine level in explanation tissue and body is closely bound up.And as renal function mark, the variation of understanding creatinine content in zebra fish tissue has important indicative significance to grasping renal function state.
Summary of the invention
The present invention is directed to now methodical deficiency, provide a kind of screening to there is the method for protecting or improving renal function reactive compound.
Summary of the invention
The present invention can cause the compound of kidney injury and testing compound acting in conjunction in zebrafish embryo; or the first embryo after zebrafish embryo causes kidney injury and then damages with testing compound processing by renal toxicity compound effects; utilize in embryonic tissue creatinine content change as detecting the index of renal function; whether analyze testing compound has protection or improves renal function activity; set up a kind ofly have fast, the screening technique of the reactive compound of reliable, high flux feature, for the research and development of kidney diseases medicine provide lead compound.
Detailed Description Of The Invention
Technical scheme of the present invention is as follows:
Screening has protection or improves a method for renal function reactive compound, comprises the following steps:
(1) zebrafish embryo of growing 3～6 days is put into the cultivation aqueous solution containing known renal toxicity compound, cultivate 20～28h, then remove dead embryo, clean embryo's remained on surface liquid with pure water, make damage group embryo; Adopt the cultivation water that does not add known renal toxicity compound to cultivate under the same conditions simultaneously, make normal group embryo; The zebrafish embryo of growing 3～6 days is put into the cultivation aqueous solution containing same known renal toxicity compound and testing compound, cultivate 20～28h, then remove dead embryo, clean embryo's remained on surface liquid with pure water, make treatment group embryo;
Or:
The zebrafish embryo of growing 3～6 days is put into the cultivation aqueous solution containing known renal toxicity compound, cultivate 20～28h, with the clean embryo's remained on surface liquid of pure water, then embryo is divided into two groups, one group moves in cultivation water, another group moves in the cultivation aqueous solution that contains testing compound, two groups of embryos are continued to cultivate 24h, remove dead embryo, with the clean embryo's remained on surface liquid of pure water, cultivate one group that water planting supports and be designated as damage group embryo, a group of cultivating of the cultivation aqueous solution that contains testing compound is designated as treatment group embryo; Adopt and cultivate the foster 44～52h h of water planting, make normal group embryo;
(2) the every group of embryo who makes to step (1) adds the inner mark solution of equal volume, extract polar compound wherein through homogenate, layering, extract sample after nitrogen dries up, add pure water to dissolve, then cross 0.45 μ m polyethersulfone water filter membrane, filtrate is injected to LC-MS/MS, carry out compartment analysis; Then respectively creatinine standard solution and interior mark standard solution are injected to LC-MS/MS, detect under the same conditions;
(3) according to following formula:
C testing sample=C standard items × AA testing sample/AA Standard product
In formula: C is for extracting sample concentration, and AA is chromatographic peak area;
Calculate creatinine and interior mark content in testing sample, then calculate the each group of creatinine content in embryo's sample according to interior mark extraction ratio;
(4) respectively organize creatinine content in embryonic tissue and represent with means standard deviation, adopt the relatively conspicuousness of group difference of independent sample t method of inspection analysis;
When the creatinine content in damage group embryonic tissue is higher than the creatinine content in normal group embryonic tissue, and reach level of significance (P<0.05) statistically, successfully cause embryo's kidney function damage;
Occur under damage prerequisite at embryo's renal function; when creatinine content in treatment group embryonic tissue is lower than the creatinine content in damage group embryonic tissue; and reach level of significance (P<0.05) statistically, testing compound has the activity of protecting or improving renal function.
Preferred according to the present invention, zebrafish embryo condition of culture is in described step (1): under 28 DEG C, 14h illumination/10h dark condition, cultivate.
Preferred according to the present invention, described step (1) middle kidney toxic chemical is aristolochic acid, and concentration is 0.5～4 μ g/mL.
Preferred according to the present invention, in described step (1), the each group of embryo who makes is no more than 7 days apart from fertilization time.Inventor finds by great many of experiments, and fertilization time exceedes the embryo after 7 days, owing to being subject to multiple disturbing factor impact, can cause creatinine Detection accuracy to reduce.
Preferred according to the present invention, in described step (1), described zebrafish embryo is that wild type or AB are zebrafish embryo.
Further preferred according to the present invention, above-mentioned zebrafish embryo is for fertilization is more than 3 days, and wild type or AB that kidney has completed growth are zebrafish embryo.
Preferred according to the present invention, in described step (1), the cultivation water that contains testing compound is to add in water mixing of testing compound or testing compound and cosolvent cultivating.
The object of above-mentioned cosolvent is to promote the dissolving of testing compound, cosolvent concentration range planted agent used not to the effect of embryogenesis Toxicity of Kidney.
The toxicity that the concentration of above-mentioned testing compound produces according to different testing compounds is different and not identical, and those skilled in the art can adjust selection voluntarily according to experiment situation.
Further preferred according to the present invention, above-mentioned cultivation water component is:
NaCl 5mmol/L, KCl 0.17mmol/L, CaCl
20.4mmol/L, MgSO
40.16mmol/L, deionized water preparation.
Creatinine content in described embryonic tissue is the quality of creatinine contained in each fixed number object embryonic tissue.Creatinine structural formula is as follows:
Preferred according to the present invention, in described step (1), every group of embryo's sum is not less than 30 pieces.Further preferred, every group of embryo adds up to 50～150 pieces.
Preferred according to the present invention, in described step (2), the internal standard compound matter adding in embryo's sample is Cimetidine standard solution.Cimetidine structural formula is as follows:
Further preferred according to the present invention, the addition of above-mentioned Cimetidine standard items is that every 50 embryos add 3～10ng.
Preferred according to the present invention, in described step (2), extraction polar compound is water: methyl alcohol: chloroform is (60～70) by volume: (75～85): the mixed liquor that mix (75～85) extracts.
Beneficial effect:
1, the present invention sets up the new method that a kind of screening based on model organism zebra fish has protection and improves renal function reactive compound, first utilize renal toxicity compound (modeling compound) to cause zebrafish embryo renal dysfunction, then process the zebrafish embryo after damage with testing compound, improve the activity of average evaluation compound according to zebrafish embryo renal function; Or renal toxicity compound and testing compound are processed to zebrafish embryo simultaneously, then according to the activity of zebrafish embryo renal function average evaluation compound.The method is quick, accurate, easy.
2, the present invention is using the creatinine level in embryonic tissue as the foundation that detects renal function, establish the quantitatively evaluating index of the renal function damage that is applicable to zebrafish embryo, compared with existing zebrafish embryo renal function detection method, have more specificity and representativeness.
3, model organism zebra fish embryo used in the present invention; it had both had advantages of that cell in vitro strain can rapid screening, had again the advantage of checking in living animal body, utilized zebrafish embryo to carry out the screening of extensive reactive compound; contribute to improve screening effeciency, reduce experimental cost.
4, the present invention selects and grows 3～6 days, and the zebrafish embryo that kidney has completed growth is model, strictly limits each group of embryo and is no more than 7 days apart from fertilization time, has improved the accuracy of testing result.
Brief description of the drawings
Relatively histogram of creatinine content after Fig. 1, aristolochic acid A and Rhein processing 24h in zebrafish embryo (5dpf) tissue;
In figure: *: with normal group comparison, P<0.05; #: with the comparison of damage group, P<0.05;
Zebrafish embryo (6dpf) renal tissue section (HE dyeing) photo after Fig. 2, aristolochic acid A and Rhein (20 μ g/mL) processing 24h;
In figure: gl, glomerulus, pt, pronephric duct; N: notochord.
Fig. 3, Puerarin are to relatively histogram of creatinine content in renal function damage zebrafish embryo tissue;
In figure: *: with normal group comparison, P<0.05; #: with the comparison of damage group, P<0.05;
Fig. 4, aristolochic acid A and Puerarin (10 μ g/mL) are processed rear zebrafish embryo (5dpf) renal tissue section (HE dyeing) photo;
In figure: gl, glomerulus, pt, pronephric duct; N: notochord.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is further elaborated, but institute of the present invention protection domain is not limited to this.
Animal used as test: wild type used or AB are healthy zebra fish, for common commercially available prod, this area, at 28 DEG C, cultivates under 14h illumination/10h dark condition.Every day is in 9:30 (am) and twice fairy shrimp of 4:30 (pm) feeding.Ovulation was put in adult fish to lay eggs in cylinder than 2:1 by male and female the day before yesterday, and the middle dividing plate of placing, is placed in dark surrounds, pump dividing plate before bright light next day, and light stimulation makes its ovulation, after half an hour, adult fish is pulled out, ovulation period was controlled in half an hour, to reduce the difference of development time between embryo.Collect embryonated egg, rinse after 3 times with the new water of cultivating, be placed in 28 DEG C of incubators, keep 14h illumination/10h dark cycle to cultivate, middle every 24h 1/3 water of exchanging treaties, and sucking-off dead embryo in time.
Reagent preparation: creatinine standard items used, Cimetidine standard items, aristolochic acid A standard items, Rhein standard items and Puerarin standard items are all purchased from National Institute for Food and Drugs Control, creatinine and Cimetidine dissolve and are mixed with 10 μ g/mL creatinine standard solutions and 4 μ g/mL Cimetidine standard solutions with pure water respectively, aristolochic acid A, Rhein and Puerarin are mixed with 2mg/mL aristolochic acid A liquid storage with dmso solution respectively, the Puerarin liquid storage of 5mg/mL Rhein liquid storage and 5mg/mL, all samples liquid storage is put in 4 DEG C of preservations, when experiment, be diluted to desired concn with cultivation water.
Cultivation water component is as follows:
NaCl 5mmol/L, KCl 0.17mmol/L, CaCl2 0.4mmol/L, MgSO4 0.16mmol/L, deionized water preparation.
Embodiment 1: the protective effect of Rhein to zebrafish embryo renal function
Select 24 porocyte culture plates, in hole, add appropriate aristolochic acid A liquid storage, be diluted to 2 μ g/mL as damage group with cultivation water; In hole, add successively appropriate aristolochic acid A liquid storage and Rhein liquid storage, add and cultivate water dilution, obtain the mixed solution containing 2 μ g/mL aristolochic acid As and variable concentrations Rhein, the concentration of Rhein is followed successively by 5,10,20 μ g/mL, respectively as treatment group 1, treatment group 2 and treatment group 3; In hole, add the cultivation aqueous solution that contains 0.5% (v/v) DMSO (dimethyl sulfoxide (DMSO)) as normal group.Each hole solution final volume is 2mL, careful solution in all samples hole piping and druming is mixed with suction pipe.The random zebrafish embryo of growing 5 days sizes of drawing, moves in above-mentioned sample hole 15, every hole embryo, every group of 4 holes.Add a cover after sealing, put into 28 DEG C of incubators, under 14h illumination/10h dark, hatch 24h.
After collection and treatment, each group embryo, removes dead embryo, and makes between each group embryo number identical.Embryo, with after pure water rinsing 3 times, is moved in 1.5mL Eppendorf pipe, and moisture exhausts as far as possible, add the pre-cold methanol of 400 μ L, 124 μ L precooling pure waters, 1 μ L Cimetidine inner mark solution, homogenate 2min then adds 400 μ L chloroforms, 200 μ L precooling pure waters in every pipe, vortex vibration 60S, stratification, then 4 DEG C, centrifugal 5min under 10000 × g condition, carefully draws supernatant liquid and moves to new 1.5mL Eppendorf pipe, nitrogen dries up, and dries up sample and is put in-70 DEG C of preservations.
Adopt SHIMADZU VP-ODS post (250mm × 4.6mm, 3 μ m), analysis time 12min, sample size 5 μ L.Elution requirement: mobile phase A (methyl alcohol) and Mobile phase B (pure water) are carried out gradient elution (in table 1), flow rate of mobile phase: 1.0mL/min according to different proportion.
Mass spectrum condition: electric spray ion source polarity: spray voltage: 3000V; Gasification temperature: 350 DEG C; Sheath air pressure: 60arb; Assist gas pressure: 30arb; Ion transfer tube temperature: 300 DEG C; Collision gas (Ar) 1.5mTorr; Scan pattern SRM.
Table 1 utilizes LC-MS/MS to detect creatinine mobile phase system used in zebrafish embryo tissue
Get nitrogen and dry up sample and add 1mL pure water fully to dissolve, cross 0.45 μ m polyethersulfone water filter membrane, get 5 μ L filtrates and inject LC-MS/MS and carry out compartment analysis by above-mentioned condition.
Respectively creatinine standard solution and interior mark standard solution are diluted to (creatinine standard items are diluted to concentration 100ng/mL, interior mark standard items are diluted to concentration 15ng/mL), respectively get 5 μ L and inject LC-MS/MS, under identical chromatographic condition, measure, utilize external standard method to calculate creatinine and the interior mark content in testing sample, and according to the creatinine content in interior mark extraction ratio calculation sample; Computing formula:
C
testing sample=C
standard items× AA
testing sample/ AA
standard items
In formula: C, extraction sample concentration, AA, chromatographic peak area.
Table 2 LC-MS/MS detects the creatinine content in different disposal group zebrafish embryo (6dpf)
N: every group of Duplicate Samples number; A: with normal group comparison, P<0.05; B: with the comparison of damage group, P<0.05.
Result shows, 5dpf (growing 5 days) zebrafish embryo is after 2 μ g/mL aristolochic acid As are processed 24h, creatinine content in embryonic tissue is 98.74 ± 7.5ng/50 embryo, higher than normal group (72.91 ± 8.1ng/50 embryo) (table 2), and there is significant difference (P<0.05) (Fig. 1).From renal tissue section result, find that short texture, cell arrangement disorder and Cystic changes (as shown in Figure 2) appear in aristolochic acid A processed group embryo renal glomerulus, show that renal tissue structure suffers damage, 2 μ g/mL aristolochic acid As cause kidney injury.
When adding aristolochic acid A, add the compound of variable concentrations, after co-treatment embryo 24h, creatinine content in embryonic tissue is respectively 92.63 ± 5.5ng/50 embryo (treatment group 1), 84.43 ± 4.8ng/50 embryo (treatment group 2), 81.24 ± 6.3ng/50 embryo (treatment group 3), all there are rising, wherein no significant difference between treatment group 3 and normal group with normal group ratio.With damage group ratio, three treatment group creatinine values all have reduction, wherein between treatment group 2 and treatment group 3 and damage group, have significant difference (Fig. 1).Renal tissue section shows; treatment group (treatment group 2; 3) there is the abnormal change such as short texture, cell arrangement disorder in embryo's renal glomerulus; but from scope and degree, be obviously lighter than damage group (as shown in Figure 2); illustrate in adding aristolochic acid to process and add compound; can alleviate the damage of the renal function that aristolochic acid causes; improve glomerulus CrCl; draw thus, when 10 μ g/mL, 20 μ g/mL concentration, Rhein has prolection to zebrafish embryo renal function.This conforms to general knowledge well known in the prior art, therefore can prove the accuracy of this method.
Embodiment 2: the therapeutic action of Puerarin to zebrafish embryo renal dysfunction
Select 6 porocyte culture plates, with cultivation water, aristolochic acid A liquid storage is diluted to the working solution of 4 μ g/mL, join in 4 sample holes, 6mL/ hole, the random zebrafish embryo of growing 3 days sizes of drawing, puts into aristolochic acid A working solution, 60, every hole embryo, add a cover after sealing, put into 28 DEG C of incubators, under 14h illumination/10h dark, hatch 24h.Then collecting all embryos cleans after 3 times with cultivating water, be divided at random four groups, one group moves into and cultivates in aqueous solution as damage group, and another three groups of embryos move into respectively in the Puerarin solution of 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, obtain medical treatment group 1, treatment group 2, treatment group 3.Under same culture conditions, the cultivation water planting of zebrafish embryo containing 0.4% (v/v) DMSO (dimethyl sulfoxide (DMSO)) of growing 3 days sizes supported to 48h as normal group.
After collection and treatment, each group embryo, removes dead embryo, and makes between each group embryo number identical.Embryo, with after pure water rinsing 5 times, is moved in 1.5mL Eppendorf pipe, and moisture exhausts as far as possible, add the pre-cold methanol of 400 μ L, 124 μ L precooling pure waters, 1 μ L Cimetidine inner mark solution, homogenate 2min then adds 400 μ L, 200 μ L precooling pure waters in every pipe, vortex vibration 60S, stratification, then 4 DEG C, centrifugal 5min under 10000 × g condition, carefully draws supernatant liquid and moves to new 1.5mLEppendorf pipe, nitrogen dries up, and dries up sample and is put in-70 DEG C of preservations.
Adopt SHIMADZU VP-ODS post (250mm × 4.6mm, 3 μ m), analysis time 12min, sample size 5 μ L.Elution requirement: mobile phase A (methyl alcohol) and Mobile phase B (pure water) are carried out gradient elution according to different proportion, and condition is as shown in table 1, flow rate of mobile phase: 1.0mL/min.Mass spectrum condition: electric spray ion source polarity: spray voltage: 3000V; Gasification temperature: 350 DEG C; Sheath air pressure: 60arb; Assist gas pressure: 30arb; Ion transfer tube temperature: 300 DEG C; Collision gas (Ar) 1.5mTorr; Scan pattern SRM.
Get nitrogen and dry up sample and add 1mL pure water fully to dissolve, cross 0.45 μ m polyethersulfone water filter membrane, get 5 μ L filtrates and inject LC-MS/MS and carry out compartment analysis by above-mentioned condition.
Respectively creatinine standard solution and interior mark standard solution are diluted to (creatinine standard items are diluted to concentration: 100ng/mL, interior mark standard items are diluted to concentration: 15ng/mL), respectively get 5 μ L and inject LC-MS/MS, under identical chromatographic condition, measure, utilize external standard method to calculate creatinine and the interior mark content in testing sample, and according to the creatinine content in interior mark extraction ratio calculation sample; Computing formula:
C
testing sample=C
standard items× AA
testing sample/ AA
standard items
In formula: C, extraction sample concentration, AA, chromatographic peak area.
Table 3 LC-MS/MS detects the creatinine content in different disposal group zebrafish embryo (5dpf)
N: every group of Duplicate Samples number; A: with normal group comparison, P<0.05; B: with the comparison of damage group, P<0.05.
Result shows, creatinine content in damage group embryonic tissue is 78.79 ± 4.32ng/50 embryo, higher than normal group 47.5 ± 5.61ng/50 embryo's (table 3), and there is significant difference (P<0.05) (Fig. 3), renal tissue section result shows, 4 μ g/mL aristolochic acid A processed group embryo's renal glomerulus and pronephric duct all occur that textural anomaly changes, there is short texture and cystic dilatation performance (as shown in Figure 4), show that renal tissue structure suffers damage.
Creatinine content in treatment group 1 embryonic tissue is 72.91 ± 5.33, organizes low height than damage, but there was no significant difference.Creatinine content in treatment group 2 and treatment group 3 embryonic tissues is than damage group low (table 3), and has significant difference, the more equal no significant difference of both and normal group (Fig. 3).The demonstration of pathological section result, the infringement for the treatment of group embryo's kidney institutional framework is obviously lighter than damage group (as shown in Figure 4).
Process 24h by presentation of results aristolochic acid and can damage embryo's renal function and institutional framework, cause creatinine level in tissue to raise, even if remove after aristolochic acid, Renal Structure and dysfunction still exist.Process the embryo after damage with Puerarin, can promote the recovery of its structure and function.Illustrate that Puerarin has the activity for the treatment of kidney injury in the time of 5 μ g/mL, 10 μ g/mL concentration.This conforms to general knowledge well known in the prior art, therefore can prove the accuracy of this method.
Claims (10)
1. screening has the method protecting or improve renal function reactive compound, it is characterized in that, comprises the following steps:
(1) zebrafish embryo of growing 3～6 days is put into the cultivation aqueous solution containing known renal toxicity compound, cultivate 20～28h, then remove dead embryo, clean embryo's remained on surface liquid with pure water, make damage group embryo; Adopt the cultivation water that does not add known renal toxicity compound to cultivate under the same conditions simultaneously, make normal group embryo; The zebrafish embryo of growing 3～6 days is put into the cultivation aqueous solution containing same known renal toxicity compound and testing compound, cultivate 20～28h, then remove dead embryo, clean embryo's remained on surface liquid with pure water, make treatment group embryo;
Or:
The zebrafish embryo of growing 3～6 days is put into the cultivation aqueous solution containing known renal toxicity compound, cultivate 20～28h, with the clean embryo's remained on surface liquid of pure water, then embryo is divided into two groups, one group moves in cultivation water, another group moves in the cultivation aqueous solution that contains testing compound, two groups of embryos are continued to cultivate 24h, remove dead embryo, with the clean embryo's remained on surface liquid of pure water, cultivate one group that water planting supports and be designated as damage group embryo, a group of cultivating of the cultivation aqueous solution that contains testing compound is designated as treatment group embryo; Adopt and cultivate the foster 44～52h h of water planting, make normal group embryo;
(2) the every group of embryo who makes to step (1) adds the inner mark solution of equal volume, extract polar compound wherein through homogenate, layering, extract sample after nitrogen dries up, add pure water to dissolve, then cross 0.45 μ m polyethersulfone water filter membrane, filtrate is injected to LC-MS/MS, carry out compartment analysis; Then respectively creatinine standard solution and interior mark standard solution are injected to LC-MS/MS, detect under the same conditions;
(3) according to following formula:
C testing sample=C standard items × AA testing sample/AA Standard product
In formula: C is for extracting sample concentration, and AA is chromatographic peak area;
Calculate creatinine and interior mark content in testing sample, then calculate the each group of creatinine content in embryo's sample according to interior mark extraction ratio;
(4) respectively organize creatinine content in embryonic tissue and represent with means standard deviation, adopt the relatively conspicuousness of group difference of independent sample t method of inspection analysis;
When the creatinine content in damage group embryonic tissue is higher than the creatinine content in normal group embryonic tissue, and reach level of significance statistically, successfully cause embryo's kidney function damage;
Occur, under damage prerequisite, when creatinine content in treatment group embryonic tissue is lower than the creatinine content in damage group embryonic tissue, and to reach level of significance statistically at embryo's renal function, testing compound has the activity of protecting or improving renal function.
2. the method for claim 1, is characterized in that, zebrafish embryo condition of culture is in described step (1): under 28 DEG C, 14h illumination/10h dark condition, cultivate.
3. the method for claim 1, is characterized in that, described step (1) middle kidney toxic chemical is aristolochic acid, and concentration is 0.5～4 μ g/mL.
4. the method for claim 1, is characterized in that, in described step (1), the each group of embryo who makes is no more than 7 days apart from fertilization time.
5. method as claimed in claim 4, is characterized in that, in described step (1), described zebrafish embryo is that wild type or AB are zebrafish embryo.
6. the method for claim 1, is characterized in that, described zebrafish embryo is that wild type or the AB that the above and kidney of fertilization 3 days has completed growth is zebrafish embryo.
7. the method for claim 1, is characterized in that, in described step (1), the cultivation water that contains testing compound is to add in water mixing of testing compound or testing compound and cosolvent cultivating.
8. method as claimed in claim 7, is characterized in that, described cultivation water component is:
NaCl 5mmol/L, KCl 0.17mmol/L, CaCl
20.4mmol/L, MgSO
40.16mmol/L, deionized water preparation.
9. the method for claim 1, is characterized in that, in described step (1), every group of embryo's sum is not less than 30 pieces; Further preferred, every group of embryo adds up to 50～150 pieces.
10. the method for claim 1, is characterized in that, in described step (2), the internal standard compound matter adding in embryo's sample is Cimetidine standard solution; Further preferred according to the present invention, the addition of above-mentioned Cimetidine standard items is that every 50 embryos add 3～10ng;
Preferred according to the present invention, in described step (2), extraction polar compound is water: methyl alcohol: chloroform is (60～70) by volume: (75～85): the mixed liquor that mix (75～85) extracts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410377736.0A CN104142384B (en) | 2014-08-01 | 2014-08-01 | It is a kind of to screen with the method for protecting or improving renal function reactive compound |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410377736.0A CN104142384B (en) | 2014-08-01 | 2014-08-01 | It is a kind of to screen with the method for protecting or improving renal function reactive compound |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104142384A true CN104142384A (en) | 2014-11-12 |
| CN104142384B CN104142384B (en) | 2018-03-09 |
Family
ID=51851617
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410377736.0A Active CN104142384B (en) | 2014-08-01 | 2014-08-01 | It is a kind of to screen with the method for protecting or improving renal function reactive compound |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104142384B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105169415A (en) * | 2015-08-10 | 2015-12-23 | 山东省科学院生物研究所 | Method for screening compounds having activity of protecting liver function of zebra fishes |
| CN106370833A (en) * | 2016-08-16 | 2017-02-01 | 山东省科学院生物研究所 | Method for evaluating effect of compound on angiogenesis in pathological state |
| CN107890466A (en) * | 2017-12-05 | 2018-04-10 | 赵腾骅 | A kind of chromene lactone is used for the purposes that for aristolochic acid and the Chinese medicine containing aristolochic acid is attenuated |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1439364A (en) * | 2003-03-26 | 2003-09-03 | 浙江大学 | Protective composite amino acid composition for kidney and its use |
| CN103616489A (en) * | 2013-11-28 | 2014-03-05 | 河北科技大学 | Method for testing typical antibiotics wastewater toxicity by using zebra fishes |
-
2014
- 2014-08-01 CN CN201410377736.0A patent/CN104142384B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1439364A (en) * | 2003-03-26 | 2003-09-03 | 浙江大学 | Protective composite amino acid composition for kidney and its use |
| CN103616489A (en) * | 2013-11-28 | 2014-03-05 | 河北科技大学 | Method for testing typical antibiotics wastewater toxicity by using zebra fishes |
Non-Patent Citations (7)
| Title |
|---|
| DUAN ZHENG-HUA: "A Metabonomic Investigation of Zebrafish exposed to Bisphenol A", 《BIOINFORMATICS AND BIOMEDICAL ENGINEERING》 * |
| 刘桂香: "硫代硫酸钠对顺铂所致肾毒性中和作用的实验研究", 《中国职业医学》 * |
| 杨敬华: "BSO和维生素C、E对汞毒性影响的实验研究", 《中国优秀博硕士学位论文全文数据库（硕士） 医药卫生科技辑》 * |
| 王筝: "柴苓汤对环孢素A慢性肾毒性大鼠RAS系统的作用", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 王雪: "斑马鱼胚胎在肾毒性损伤研究中的应用进展", 《中国药理学与毒理学杂志》 * |
| 程根阳: "罗格列酮防治环孢素A所致大鼠肝肾毒性的分子机制研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 赵怡蕊: "黄芪与广防已优化组合对大鼠急性肾毒性保护作用的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105169415A (en) * | 2015-08-10 | 2015-12-23 | 山东省科学院生物研究所 | Method for screening compounds having activity of protecting liver function of zebra fishes |
| CN105169415B (en) * | 2015-08-10 | 2018-07-24 | 山东省科学院生物研究所 | A method of screening has protection zebra fish liver function reactive compound |
| CN106370833A (en) * | 2016-08-16 | 2017-02-01 | 山东省科学院生物研究所 | Method for evaluating effect of compound on angiogenesis in pathological state |
| CN106370833B (en) * | 2016-08-16 | 2018-04-24 | 山东省科学院生物研究所 | It is a kind of to be used to evaluate method of the compound to angiogenesis function under pathological state |
| CN107890466A (en) * | 2017-12-05 | 2018-04-10 | 赵腾骅 | A kind of chromene lactone is used for the purposes that for aristolochic acid and the Chinese medicine containing aristolochic acid is attenuated |
| CN107890466B (en) * | 2017-12-05 | 2018-12-21 | 东阳市新意工业产品设计有限公司 | A kind of chromene lactone is used for as aristolochic acid and the purposes of the attenuation of the Chinese medicine containing aristolochic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104142384B (en) | 2018-03-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109633140B (en) | Method for evaluating neurodevelopment toxicity of perfluorinated compounds by using zebra fish | |
| CN101579451B (en) | Chicken coccidia powder and preparation method thereof | |
| CN105115951B (en) | A kind of method of Fast Evaluation compound to zebra fish liver function damaging action | |
| CN101810866A (en) | New method for screening anti-liver injury medicament by using model organism zebra fish | |
| CN104122355B (en) | Method for evaluating kidney toxicity of compounds through detecting contents of creatinine in zebra fish tissues | |
| CN104278077B (en) | A kind of method of high-flux fast screening Macrobrachium rosenbergii immunostimulant | |
| CN102353663A (en) | Method for carrying out quantitative evaluation to compound hepatotoxicity by using zebra fish | |
| CN104142384A (en) | Method for screening active compounds capable of protecting or improving renal functions | |
| CN106035144A (en) | Disease resistance evaluation method of cultured shrimps and crabs | |
| CN102023207B (en) | Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof | |
| CN1969955A (en) | Health-preserving capsule of edible fungus, preparation method and use thereof | |
| CN101862366B (en) | Method for extracting medicament for treating allergic colitis from Alhagi sparsifolia plant and medicament and application thereof | |
| CN101878756B (en) | Method for storing monochamus alternatus hope sample and method for quickly detecting bursaphelenchus xylophilus therein | |
| JP2013145250A (en) | Toxicity testing method | |
| CN104297222A (en) | Zebrafish embryo alcoholic liver detecting model and construction method and application of zebrafish embryo alcoholic liver detecting model | |
| CN104922268B (en) | A kind of method that active material is extracted from natural plants | |
| Altaee | In vivo toxicity study of nerium oleander's leaves and flowers aqueous extracts in mice (Cytogenetic, Biochemical and hematological Study) | |
| CN106924297A (en) | A kind of Lachnum intracellular melanin as acute liver damage medicine purposes | |
| CN106929560A (en) | A kind of method for determining chemicals development toxicity using tropical Xenopus Embryo | |
| Patnaik | Biochemical alterations induced by sevin in Clarias batrachus | |
| CN105381024A (en) | Pharmaceutical composition for treating chicken coccidiosis and preparation method thereof | |
| CN1839922A (en) | Traditional Chinese medicine preparation for treating leukoderma, psoriasis and its preparation method | |
| CN103961339B (en) | The application of new nuzhenide in adriamycin myocardial toxicity and the pharmaceutical composition being main active with new nuzhenide | |
| CN103961338B (en) | The application of hydroxytyrosol in adriamycin myocardial toxicity and take hydroxytyrosol as the pharmaceutical composition of main active | |
| CN113495125A (en) | Method for evaluating safety of grain crops by using zebra fish |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |