CN111307982A - In-vitro high-throughput screening platform for effect of drug candidate on intestinal flora - Google Patents
In-vitro high-throughput screening platform for effect of drug candidate on intestinal flora Download PDFInfo
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Abstract
The invention discloses an in vitro high-flux screening platform for the effect of a drug candidate on intestinal flora, which is characterized in that a plurality of representative intestinal strains are selected according to the characteristics of the drug candidate and the intestinal flora of target people to simulate a simplified human intestinal microbial environment; the method comprises the steps of diluting the intestinal canal strain in a microbial culture medium, placing the diluted intestinal canal strain in an integrated pore plate, keeping the initial concentration of a bacterial suspension of the intestinal canal strain between 0.2 and 1.0, and realizing high-throughput screening of the action effect of a target drug candidate and the intestinal canal strain according to growth phenotype detection of the intestinal canal strain and microbial metabolite detection of a target compound.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to an in-vitro high-throughput screening platform for the effect of a drug candidate on intestinal flora.
Background
In past studies, in vitro screening methods based on in vitro cell culture and target-based drug activity screening (including enzymatic targets, GPCR targets, ion channel targets, nuclear receptor targets, etc.) have been commonly used for new drugs and drug candidates. However, in recent years, with the progress of studies on intestinal microorganisms, it has been found that a considerable part of drugs act on the body indirectly by interacting with the intestinal flora of the human body, in addition to directly interacting with the host by being absorbed into the blood or acting on the target of host cells. In recent researches, two groups in succession respectively prove that the intestinal flora participates in the metabolism and the efficacy of various medicines in Nature and Science journal texts. At present, animal models are mostly adopted to evaluate the interaction between the drug and the intestinal flora, and a high-throughput rapid screening method similar to an in vitro cell screening platform is lacked. Therefore, we developed here an in vitro high throughput screening platform for drug-gut flora effect based on pure culture system of gut strains.
Disclosure of Invention
The invention aims to provide an in-vitro high-throughput screening platform for the effect of a drug candidate on intestinal flora, which aims to solve the existing problems.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention comprises the following steps:
A. selecting a plurality of representative intestinal strains to simulate a simplified human intestinal microbial environment according to the drug candidates and the characteristics of the intestinal flora of the target population;
b, diluting the intestinal tract strains in a microbial culture medium, and then placing the diluted intestinal tract strains in an integrated pore plate, wherein the initial concentration of the bacterial suspension of the intestinal tract strains is kept between 0.2 and 1.0;
C. respectively dripping the target drug candidates into the integrated pore plate through a microfluidic high-flux liquid distribution system, and then carrying out anaerobic culture on the intestinal strain in a continuous gas supply device;
D. culturing for 12-72 hours, and performing growth phenotype detection and metabolite detection based on optical parameters on the intestinal bacteria;
E. and according to the growth phenotype detection of the intestinal bacteria and the microbial metabolite detection of the target compound, the interaction effect of the drug candidate and the intestinal microorganisms is screened at high flux for the interaction effect of the target drug candidate and the intestinal bacteria.
Further, the metabolite detection reflects the effect of different drug candidates on the gut species metabolite detection activity through the gut species biomass change in the integrated well plate.
Specifically, the metabolite detection method can be a chromatography method, a mass spectrometry method or a chromatography-mass spectrometry combined method.
Further, the growth parameters include, but are not limited to, absorbance values in the visible range and fluorescence absorbance values.
Specifically, the method for selecting the intestinal bacteria comprises the steps of determining a target population targeted by the screening according to a candidate drug to be screened, extracting characteristic information such as relative abundance, occurrence frequency and remarkable difference degree of each bacteria according to an analysis result of the characteristics of the target population, and determining and selecting the number and the type of specific representative intestinal bacteria by taking the information as a standard so as to construct the integrated porous plate for bearing the intestinal bacteria.
Further, the growth phenotype detection judges whether the drug candidate affects the growth phenotype of the intestinal bacteria by calculating the absorbance value ratio of the plurality of intestinal bacteria processing groups added with the drug candidate and the corresponding blank control group.
Specifically, when the ratio of the absorbance values is <1.0, it is judged that the bacterial species can metabolize and utilize the compound, and when the ratio > <1.0, it is judged that the bacterial species cannot metabolize and utilize the compound.
Compared with the prior art, the invention has the beneficial effects that:
the method overcomes the defects of high cost, long period, low flux and poor repeatability of the method for evaluating the interaction of the drug and the intestinal flora based on the animal model through the high-flux screening method, has quite high flux, stability and repeatability, provides a feasible screening platform for the active drug with the action effect of the intestinal flora, avoids the shortage of funds and blindness of direction selection in the research of the prior new drug, and reduces the adverse reaction of the active drug caused by the pharmacological action generated by clinical medication.
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FIG. 1 is a flow chart of an in vitro high throughput screening platform for the effect of drug candidates on gut flora according to the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
A. Selecting a plurality of representative intestinal strains to simulate a simplified human intestinal microbial environment according to the drug candidates and the characteristics of the intestinal flora of the target population;
b, diluting the intestinal tract strains in a microbial culture medium, and then placing the diluted intestinal tract strains in an integrated pore plate, wherein the initial concentration of the bacterial suspension of the intestinal tract strains is kept between 0.2 and 1.0;
C. respectively dripping the target drug candidates into the integrated pore plate through a microfluidic high-flux liquid distribution system, and then carrying out anaerobic culture on the intestinal strain in a continuous gas supply device;
D. culturing for 12-72 hours, and performing growth phenotype detection based on optical parameters and metabolite detection based on chromatography and mass spectrometry on the intestinal bacterial strain;
E. and according to the growth phenotype detection of the intestinal bacteria and the microbial metabolite detection of the target compound, the interaction effect of the drug candidate and the intestinal microorganisms is screened at high flux for the interaction effect of the target drug candidate and the intestinal bacteria.
Example one
In this implementationIn the case, Inosine (Inosine) is selected as a medicament to be screened, and Inosine is also called hypoxanthine nucleoside, is a biological fermentation product, can be used for the adjuvant treatment of acute and chronic hepatitis, leucopenia and thrombocytopenia caused by various reasons in medical treatment, and is also used for treating heart diseases such as pulmonary heart disease and the like and treating heart or liver toxicity reaction caused by anti-schistosome medicaments. The effect of the compound on intestinal flora is unknown, and the effect of the compound on intestinal flora of people with liver injury is expected to be researched through the screening. Determining that the in vitro intestinal flora environment to be simulated is the intestinal tract of the non-alcoholic fatty liver disease crowd, and then analyzing the high-throughput sequencing data of 324 non-alcoholic fatty liver disease human intestinal flora to determine the relative abundance of strains in the crowd sample>0.1%, frequency of occurrence in all samples>50% of 22 species of the genus Enterobacter (Alisipes, Bacteroides, Blautia, Clostridium, Coprococcus, Diarister, Dorea, Dysosmobacter, Eubacterium, Novgen _2619, Flint acter, Novgen _2710, Lachnospira, Megasphaera, Parabacteraides, Phascolatobacter, Prevotella, Roseburia, Novgen _ L23, Ruminococcus, Streptococcus, Vellonella) were selected as the species of the gut for constructing an in vitro gut bacteria screening system integrated in a multi-well plate. After selecting representative strains, respectively inoculating the strains into self-improved mGMB anaerobic culture medium for seed liquid preparation, and measuring the OD value of the culture liquid at 595nm every 12 hours after inoculation until the OD value is up to595nmNo further increase in value was considered to be complete. The growth rate of the selected gut bacteria species was scored as the length of time it took for the OD value to increase to a maximum value. The scoring criteria were as follows:
according to the growth rate, the seed liquid of each strain was diluted to different concentrations (as OD) with the culture medium595nmValue characterization) was used to prepare the intestinal bacterial integrated plate, with the following dilution criteria:
and respectively adding the intestinal strain suspensions diluted to different concentrations into a multi-hole plate, wherein each strain is added into at least 3 parallel holes, and the adding amount of each hole is 200 microliters, so as to prepare the intestinal strain integrated plate. After the screening system was constructed, inosine was added sequentially to the wells of the target multi-well plate using a microfluidic based liquid separation system to a final concentration of 20 mM. Each intestinal strain is provided with 1 blank strain control group without inosine and 2 experiment groups with inosine. Then, continuously culturing the porous plate in an anaerobic culture device with continuous gas supply for 24 hours, detecting the absorbance value in an anaerobic operation platform by a small microplate reader, and calculating the ratio (OD) of each intestinal strain treatment group added with salicin to the corresponding blank control grouptreated/ODck) Judging whether the added inosine has influence on the growth phenotype of the intestinal strain, wherein the specific standard is as follows:
the evaluation score of the effect of inosine on the growth of the selected 22 intestinal strains was a, and the evaluation score of 2 strains was a, that is, inosine promoted the growth of the 2 strains (Streptococcus anguinosus, Roseburia intestinalis); 1 species (Dysosmabacter welbionis) was evaluated as C, i.e., inosine inhibited the growth of the species, and the remaining 19 were evaluated as B, i.e., had no or only a weak effect on the growth of these intestinal species.
Whether the compound has influence on representative intestinal bacteria can be judged by measuring the growth phenotype, information such as a specific mode and a molecular mechanism of the influence can be further judged by detecting metabolites, and evaluation of the action mode of the most intestinal microorganisms of the target drug candidate can be finally completed by comprehensively analyzing two results. The detailed steps of metabolite detection are as follows: taking 100 microliter of fermentation liquor from each hole of the multi-hole plate subjected to growth phenotype evaluation, centrifuging, filtering, and performing liquid chromatography-tandem mass spectrometry, wherein the liquid chromatography analysis parameters are different according to different drug candidates to be detectedParameters for inosine detection were set as: c18 column, mobile phase: a is water (containing 0.5% phosphoric acid), B is methanol; the flow rate was 0.60mL/min, and the detection wavelength (. lamda.) was 248 nm. When in analysis, a standard substance of the analytical pure inosine compound is used as a positive control, and a standard curve of inosine is established at the same time; intestinal strain pure culture solution without inosine addition was used as negative control. The initial inosine content (C) was determined by measuring the concentration of inosine in a blank sample without any addition of any enteric bacteria and with 20mM inosine added to the medium aloneInitial) Subsequently, all the treatment group samples were subjected to liquid chromatography to determine the inosine concentration (C)Remainder of). Using the residual concentration C of inosine in the fermentation liquorRemainder of(mM) and initial inosine concentration CInitialThe ratio in mM measures the mode of action of intestinal species on inosine. The specific evaluation criteria are as follows:
in the 30 intestinal strain treatment groups, the action mode scores of 2 samples are M, and the other 20 samples are N. And then, further analyzing the chromatogram of the treatment group with the score of M, wherein the two samples have a chromatographic characteristic peak which is not contained in the initial sample group of the negative control combination, the samples with the peak-off time of the characteristic peak are collected and subjected to tandem mass spectrometry, and the mass spectrometry result is analyzed by a control database, so that the compound corresponding to the characteristic peak is hypoxanthine, the metabolism of two intestinal bacteria with inosine scored as M is indicated, and the metabolite of the two intestinal bacteria is hypoxanthine.
Performing combined analysis on the growth phenotype scoring result, the drug metabolism phenotype scoring result and the metabolite analysis result to finally give an interaction screening result of the representative intestinal strain and the target drug candidate, wherein the quantitative evaluation standard is as follows:
by comprehensive assessment, the action modes of inosine and 22 representative species are respectively scored as 2A + M, 1C + N and 19B + N, namely, inosine and 2 representative species (Streptococcus anguinosus, Roseburia intestinalis) have interaction, and the action mode is to promote the growth of the intestinal representative species by participating in the metabolism of the representative species to generate hypoxanthine; in addition, inosine has direct growth inhibition effect on 1 strain (Dysosmobacter welbeiis); inosine did not interact with 19 other representative species (alitistips sp., bacteriodes fragiliss, blautiassiliensis, Clostridium symbolosum, Coprococcus eutectis, Dialister sp., Dorea sp., Eubacterium elegans, Novgen _2619, Flintibacter sp., Novgen _2710, Lachnospira sp., Megasphaera sp., parabacter discosonnis, phascobacter sp., Prevotella copri, Novgen _ L23, ruminococcus biciruses, Veillonella parvula). So far, the in vitro screening platform is used for realizing high-throughput screening and comprehensive evaluation on the action effect of a drug compound inosine and typical intestinal microorganisms of nonalcoholic fatty liver patients.
Example two:
in the embodiment, salicin is selected as the drug to be screened, and the salicin has the effects of treating cold, fever and infection, relieving arthritis pain, waist and back pain and the like. The action mechanism of the compound and intestinal flora is unknown, and the effect of the compound and intestinal flora of Asian healthy people is expected to be researched through the screening. Determining that the environment of intestinal flora to be simulated in vitro is the intestinal tract of healthy Chinese people, and then analyzing the high-throughput sequencing data of 516 intestinal flora of healthy people to determine the relative abundance of strains in a population sample>0.1%, frequency of occurrence in all samples>50% of 30 enterobacteria Alistepes, Bacteroides, Bifidobacterium, Blautia, Butyricicoccus, Clostridium, Collinsela, Coprococcus, Dyssomobacter, Eubacterium, Faecalibacillus, Intestibacter, Lactobacilli, Novgen _2710, Odoribacter, Oscilobacter, Parabacter, Phascolatobacter, Prevotella, Romboutsia, Roseburia, Ruminococcus, Streptomyces, Absysivirga, Anaetreruccus, Catenibacillus, Cellulosilyticum, Christensella, Mitsukelella were selected from each genus as an enterobacteria strain for in vitro selection of multi-well plates. At the time of selectionAfter representative strains, the strains are respectively inoculated into self-improved mGMB anaerobic culture medium for seed solution preparation, and OD values of the culture solution at 595nm are measured every 12 hours after inoculation until OD is reached595nmNo further increase in value was considered to be complete. The growth rate of the selected gut bacteria species was scored as the length of time it took for the OD value to increase to a maximum value. The scoring criteria were as follows:
according to the growth rate, the seed liquid of each strain was diluted to different concentrations (as OD) with the culture medium595nmValue characterization) was used to prepare the intestinal bacterial integrated plate, with the following dilution criteria:
and respectively adding the intestinal strain suspensions diluted to different concentrations into a multi-hole plate, wherein each strain is added into at least 3 parallel holes, and the adding amount of each hole is 200 microliters, so as to prepare the intestinal strain integrated plate. After the screening system was constructed, salicin was sequentially added to the wells of the target multi-well plate using a microfluidic based liquid separation system to a final concentration of 50 mM. Each intestinal bacterial strain is provided with 1 blank bacterial strain control group without salicin and 2 experimental groups with salicin. Then, the porous plate is continuously cultured for 48 hours in an anaerobic culture device with continuous gas supply, then the absorbance value is detected in an anaerobic operation platform by a small microplate reader, and the ratio (OD) of each intestinal strain treatment group added with salicin to the corresponding blank control group is calculatedtreated/ODck) Judging whether the added salicin has influence on the growth phenotype of the intestinal bacteria, and the specific standard is as follows:
the judgment score of the effect of the salicin on the growth of the selected 30 intestinal strains is that the evaluation score of the 13 strains is A, namely the growth of the 13 strains is promoted; the remaining 17 were judged as B, i.e. had no or only a weak effect on the growth of these intestinal species; there were no intestinal species scored as C.
Whether the compound has influence on representative intestinal bacteria can be judged by measuring the growth phenotype, information such as a specific mode and a molecular mechanism of the influence can be further judged by detecting metabolites, and evaluation of the action mode of the most intestinal microorganisms of the target drug candidate can be finally completed by comprehensively analyzing two results. The detailed steps of metabolite detection are as follows: from each well of the multi-well plate after growth phenotype evaluation, 100. mu.l of fermentation broth was subjected to a liquid chromatography-tandem mass spectrometry analysis after centrifugation, with the liquid chromatography analysis parameters set to C18 column, in methanol: water (10: 90) was used as the mobile phase, the flow rate was 1.0mL/min, and the detection wavelength was 269 nm. During analysis, a salicin compound standard substance is used as a positive control, a salicin standard curve is established, and a pure intestinal strain culture solution without salicin is used as a negative control. Firstly, measuring the salicin concentration corresponding to a blank sample which is not added with any intestinal canal strain and is only added with 50mM salicin in a culture medium as an initial salicin content value, and then sequentially carrying out liquid chromatography on all treatment group samples to measure the salicin concentration. The action mode of intestinal bacteria to salicin is measured by the ratio of the residual salicin concentration (mM) to the initial salicin concentration (mM) in the fermentation broth. The specific evaluation criteria are as follows:
the treatment groups of 30 intestinal species at this time, wherein 13 samples had an action pattern score of M and the remaining 17 were N. And then, further analyzing the chromatograms of the treatment groups with the scores of M, and if a chromatographic characteristic peak which is not existed in the initial sample group of the negative control combination is found, carrying out subsequent tandem mass spectrometry to determine which product the target candidate drug is metabolized by the strains. (in this example, no subsequent tandem mass spectrometry was performed since no new characteristic peak was generated in the chromatogram for all samples scored by M).
And (3) carrying out combined analysis on the growth phenotype scoring result and the drug metabolism phenotype scoring result to finally give a screening result of the interaction between the representative intestinal strain and the target drug candidate, wherein the quantitative evaluation standard is as follows:
by comprehensive assessment, the mode of action of salicin and 30 representative species is scored as 13 a + M and 17B + N, respectively, i.e., salicin interacts with 13 representative species (Abyssivirga sp., anaerobactuncus sp., bacteroides fragilis, Blautia maliensis, catebiaceae sp., celluliticum sp., christensella sp., Clostridium symbolosum, coprococus sp., Mitsuokella sp., paramylobacter disco, Roseburia sp., strepcoccus sp.) by participating in the metabolism of these representative species to promote the growth of these intestinal representative species; in addition, salicin did not interact with 17 other representative species (alitistips sp., Bifidobacterium bifidum, butyricucusfaecionis, collinesila _ intestinalis, dysmicrobacterium welblinis, eubacterium elegins, faecibium sp., intestinobacterium sp., Lactobacillus sp., Novgen _2710, oribacter sp., Oscillibacter sp., phascobacter sp., Prevotella copri, rombouutosia sp., ruminococcus bicirus). So far, the in vitro screening platform is used for realizing high-throughput screening and comprehensive evaluation on the action effect of a target drug candidate salicin and typical intestinal microorganisms of Asian healthy people.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. An in vitro high-throughput screening platform for the effect of drug candidates on intestinal flora, comprising the steps of:
A. selecting a plurality of representative intestinal strains to simulate a simplified human intestinal microbial environment according to the drug candidates and the characteristics of the intestinal flora of the target population;
b, diluting the intestinal tract strains in a microbial culture medium, and then placing the diluted intestinal tract strains in an integrated pore plate, wherein the initial concentration of the bacterial suspension of the intestinal tract strains is kept between 0.2 and 1.0;
C. respectively dripping the target drug candidates into the integrated pore plate through a microfluidic high-flux liquid distribution system, and then carrying out anaerobic culture on the intestinal strain in a continuous gas supply device;
D. culturing for 12-72 hours and performing phenotype detection and metabolite detection based on growth parameters on the intestinal bacteria;
E. and according to the growth phenotype detection of the intestinal bacteria and the microbial metabolite detection of the target compound, the high-throughput screening of the interaction effect of the drug candidate and the intestinal microorganisms is realized.
2. The platform of claim 1, wherein said metabolite testing reflects the effect of different drug candidates on the activity of said gut bacteria metabolite testing by altering the biomass of said gut bacteria species in an integrated well plate.
3. The platform for in vitro high-throughput screening of effect of drug candidates on gut flora according to claim 1, wherein the metabolite detection method is chromatography, mass spectrometry or a combination of chromatography and mass spectrometry.
4. The platform of claim 1, wherein the growth parameters include but are not limited to absorbance values in the visible range and fluorescence absorbance values.
5. The in vitro high-throughput screening platform for the effect of drug candidates on intestinal flora according to claim 1, wherein the selection method of intestinal bacteria species comprises the steps of determining a target population targeted by the current screening according to the drug candidates to be screened, extracting characteristic information such as relative abundance, occurrence frequency and degree of significant difference of each bacteria species according to the analysis result of the characteristics of the target population, and determining and selecting the number and the type of specific representative intestinal bacteria species according to the information as a standard to construct the integrated multi-well plate for bearing the intestinal bacteria species.
6. The in vitro high-throughput screening platform for effect of drug candidate on intestinal flora according to claim 1, wherein said growth phenotype detection determines whether said drug candidate affects the growth phenotype of intestinal flora by calculating absorbance ratio of multiple said intestinal flora treatment groups added with drug candidate and its corresponding blank control group.
7. The platform of claim 6, wherein the absorbance value ratio is <1.0, the bacterial species is determined to be able to metabolize and utilize the compound, and the ratio > is 1.0, the bacterial species is determined to be not able to metabolize and utilize the compound.
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