CN116908158A - Drug mitochondrial toxicity detection method and application thereof - Google Patents

Drug mitochondrial toxicity detection method and application thereof Download PDF

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CN116908158A
CN116908158A CN202310922547.6A CN202310922547A CN116908158A CN 116908158 A CN116908158 A CN 116908158A CN 202310922547 A CN202310922547 A CN 202310922547A CN 116908158 A CN116908158 A CN 116908158A
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mitochondrial
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高月
刘献
尚慧莹
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Academy of Military Medical Sciences AMMS of PLA
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

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Abstract

The invention discloses a drug mitochondrial toxicity detection method and application thereof, relating to the technical field of biological medicine, and the technical scheme is as follows: the method is based on a high content technology, and the mitochondrial membrane potential, the mitochondrial membrane permeability, the mitochondrial active oxygen level and the mitochondrial quality are analyzed by a mitochondrial specific fluorescent probe labeling method, so that the method for detecting the mitochondrial toxicity of the medicine is obtained. The method has the advantages of high flux, high accuracy, wide application range and the like, can efficiently detect the mitochondrial toxicity effect of the medicine, and can be applied to medicine safety evaluation and toxicology research.

Description

Drug mitochondrial toxicity detection method and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for detecting mitochondrial toxicity of a medicine and application thereof.
Background
Mitochondria are the central energy metabolism of cells and play an important role in vital activities such as cell growth, proliferation, differentiation and death. The medicine and its metabolite can cause abnormal mitochondrial structure and function, resulting in cell damage and even death, by inhibiting electron respiratory transport chain, changing mitochondrial membrane permeability, inducing mitochondrial oxidative stress, inducing mitochondrial DNA damage, etc. Mitochondrial toxicity has become an important factor in drug development failure and limited clinical application. Institutions such as the Food and Drug Administration (FDA) have clearly demanded that mitochondrial toxic effects should be included in the safety assessment of drugs.
In addition, adverse reaction events of traditional Chinese medicines represented by aristolochic acid nephropathy and the like bring about wide attention of students at home and abroad, and restrict clinical application of traditional Chinese medicine compounds and preparations thereof. Research shows that chemical components in the traditional Chinese medicine such as triptolide, aristolochic acid, aconitine and the like all cause target organ injury and organism toxic reaction due to mitochondrial toxic effect. The traditional Chinese medicine has complex and various chemical components, and the screening of mitochondrial toxic components is difficult to be realized rapidly by means of traditional animal and cell models. Therefore, there is a need to build a multidimensional, efficient mitochondrial toxicity molecular screening and evaluation model.
The high content technology uses a microplate as an experimental tool carrier at the cellular level, and utilizes a fluorescence microscope imaging and automatic picture integrated analysis system to realize detection and analysis of samples, thereby having the advantages of high throughput, high accuracy, high sensitivity and the like. At present, in the research of mitochondrial toxicity of medicines based on high content technology, the influence of medicines on single mitochondrial toxic reaction indexes is paid attention to, and the mitochondrial toxic effect of the medicines cannot be comprehensively and objectively evaluated.
Disclosure of Invention
The invention aims to provide a drug mitochondrial toxicity detection method and application thereof, and the method utilizes various types of mitochondrial specificity fluorescent probe molecules, simultaneously examines the influence of the drug on multidimensional indexes such as mitochondrial membrane potential, mitochondrial membrane permeability, mitochondrial active oxygen level, mitochondrial quality and the like, is favorable for comprehensively evaluating the mitochondrial toxicity effect of the drug, and is particularly suitable for efficient screening of mitochondrial toxic components in traditional Chinese medicines.
The technical aim of the invention is realized by the following technical scheme: the detection method takes a high content screening system as a technical means, and performs imaging analysis on living cells added with a drug to be detected based on a mitochondrial specific reaction fluorescent probe, so that the influence of the drug to be detected on the mitochondrial membrane potential, mitochondrial membrane permeability, mitochondrial active oxygen level and mitochondrial quality of the living cells is clear.
The invention is further provided with: the living cells used in the detection method are mouse normal liver cells AML12, human normal liver cells L02, human liver cancer cells HepG2, human renal tubular epithelial cells HK-2, human cardiac muscle cells AC16, rat cardiac muscle cells H9C2 and other cell types.
The invention is further provided with: in the detection method, the living cells need to be stained by using a dye before imaging analysis.
The invention is further provided with: the dye used for living cell nuclei is Hoechst33342 or DAPI.
The invention is further provided with: among the dyes, the dyes used for detecting mitochondrial membrane potential are tetramethyl rhodamine methyl ester, tetramethyl rhodamine ethyl ester, JC-1, rhodamine 123, mito-tracker red CMXRos, tetramethyl rhodamine methyl ester perchlorate or rhodamine 6G.
The invention is further provided with: the dye for detecting the mitochondrial membrane permeability is calcein AM or cobalt chloride composition.
The invention is further provided with: the dye for detecting mitochondrial active oxygen in the dye is Mitosox or MitopY1.
The invention is further provided with: among the dyes used for detecting mitochondrial mass are 10-nonylacridine orange bromide, mitoIDRed, mitotrackerGreen, mitotracker deep red633 or mitotracker red580.
The invention is further provided with: when the influence of the drug on mitochondrial membrane potential, mitochondrial membrane permeability, mitochondrial active oxygen level and mitochondrial quality is detected, the detection can be judged by comparing the average fluorescence intensity of cells of the drug group to be detected and the control group, the positive cell rate and the average fluorescence intensity of positive cells.
The invention is further provided with: when any index of mitochondrial membrane potential, mitochondrial membrane permeability, mitochondrial active oxygen level and mitochondrial quality is evaluated, the drug group to be tested has a significant difference (P < 0.05) compared with the control cells, namely, the drug is considered to have mitochondrial toxicity. And as the number of the significant difference indexes and the difference multiple are increased, the mitochondrial toxicity of the drug to be tested is considered to be enhanced.
An application of a drug mitochondrial toxicity detection method: the detection method can be used for screening mitochondrial toxic drugs, and can be used for safety evaluation and toxicology research of chemical drugs and traditional Chinese medicines.
In summary, the invention has the following beneficial effects: the invention utilizes various types of mitochondrial specificity fluorescent probe molecules, simultaneously examines the influence of the drug on multidimensional indexes such as mitochondrial membrane potential, mitochondrial membrane permeability, mitochondrial active oxygen level, mitochondrial quality and the like, is beneficial to comprehensively evaluating the mitochondrial toxicity effect of the drug, and is particularly suitable for efficiently screening mitochondrial toxic components in traditional Chinese medicines. And can be applied to safety evaluation and toxicology research of chemical medicines and traditional Chinese medicines. The method has the characteristics of high flux, high accuracy and high sensitivity, and can comprehensively and objectively evaluate the mitochondrial toxicity effect of the medicine.
Drawings
FIG. 1 high content screening of bakuchiol effects on AML12 cell mitochondrial membrane potential and statistics thereof;
FIG. 2 high content screening of bakuchiol effects on AML12 cell mitochondrial superoxide and statistics thereof;
FIG. 3 high content screening of bakuchiol effects on AML12 cell mitochondrial membrane permeability and statistics thereof;
FIG. 4 effect of doxorubicin on AC16 cell mitochondrial membrane potential and statistics thereof obtained by high content screening;
FIG. 5 high content screening of doxorubicin effect on AC16 cell mitochondrial superoxide and its statistics;
FIG. 6 high content screening of doxorubicin effect on AC16 cell mitochondrial membrane permeability and statistics thereof;
FIG. 7 is a graph showing the effect of different components of fructus Psoraleae on AML12 cell mitochondrial membrane potential and its statistics;
FIG. 8 is a graph showing the effect of different components of Psoralea corylifolia on AML12 cell mitochondrial superoxide and its statistics;
FIG. 9 shows the effect of different components of Psoralea corylifolia on the mitochondrial membrane permeability of AML12 cells and its statistics.
Detailed Description
The invention is described in further detail below with reference to fig. 1-9.
The medicinal materials, reagents, materials and instruments used in the embodiment of the invention are as follows:
psoralen (B20123), isopsoralen (B21515), bakuchiol (B20121), doxorubicin (S17092) were purchased from shanghai-derived leaf biotechnology limited. DMEM medium (11965092), DMEM/F12 medium (8122659), 0.25% edta trypsin (25200-056), PBS buffer (C10010500 BT), nuclear dye Hoechst33342 (H3570), mitoSOXRedmitochondrialsuperoxide indicator (M36008), calceinAM (C3099), tetramethylrhodamine MethylEsterPerchlorate (TMRM, T668), mitoTrackerRed (M7512) are all purchased from sameifeichi technologies (china) limited. Fetal bovine serum (04-001-1 ACS) was purchased from biologicalandustinals. Penicillin-streptomycin solution (FG 101-0) was purchased from Beijing full gold Biotechnology Co., ltd. Cobalt chloride (CoCl) 2 A 12729) was purchased from anhuizhen technologies limited.
Experimental cells: human hepatoma cells (HepG 2, CL-0103) were purchased from the Living technologies Co., ltd, and used after STR test. Human cardiomyocytes (AC 16, CL 026) were purchased from Feng Hui biosystems, and used after STR testing. Cells were seeded in 96-well cell culture plates at 37℃with 5% CO 2 Culturing under the condition.
Analysis system: high content analyzer (PerkinElmer, harmony 4.9), high content imaging system (PerkinElmer, operaPhenix).
The preparation method of the medicine comprises the following steps: accurately weighing bakuchiol, isopsoralen, psoralen and doxorubicin, respectively dissolving in DMSO, completely dissolving the medicine with an ultrasonic instrument or vortex instrument, preparing into 100mmol/L final medicinal mother liquor, and storing at-80deg.C.
Cell culture method: hepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37℃with 5% CO 2 Is cultivated in a constant temperature incubatorAnd (5) nourishing. When the cells were cultured to a density of 90%, they were passaged at 1:3 and cultured in the above medium. Cells were controlled at 3 days/generation and experimental selection was performed with cells in the logarithmic growth phase.
AC16 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37℃with 5% CO 2 Is cultured in a constant temperature incubator. When the cells were cultured to a density of 90%, they were passaged at 1:3 and cultured in the above medium. Cells were controlled at 3 days/generation and experimental selection was performed with cells in the logarithmic growth phase.
The statistical analysis method comprises the following steps: analysis was performed using graphpadprism8.0 statistical software. Experimental data are expressed in mean±sd, single-factor analysis of variance (One-wayANOVA) is used for comparison among multiple groups, and independent sample t-test is used for comparison between two groups. The difference was statistically significant in P < 0.05.
Example 1: high content screening system for analyzing type of mitochondrial toxic effect of drug liver cells
HepG2 cells were collected and counted as described above, and the cell concentration was adjusted to 10 4 Mu L/well is inoculated into a black 96-well plate and placed in an incubator for culture until the cell growth density reaches about 80%.
A blank control group and a bakuchiol administration group were set, and HepG2 cells were treated with DMEM complete medium containing 1% DMSO and bakuchiol (15. Mu. Mol/L) for 24 hours, respectively, with 5 duplicate wells per group.
The preparation method of the high content dye comprises the following steps:
(1) nucleus dye Hoechst33342: hoechst33342 is added into DMSO to prepare a mother solution of 5mg/mL, the mother solution is split into 200 mu L centrifuge tubes and stored at-80 ℃ in a dark place. Before use, the working solution is prepared into 2 mug/mL by using a culture medium without serum, and the working solution is stored at 4 ℃ in a dark place.
(2) TMRM dye: the dye was dissolved in DMSO to prepare a 10mmol/L stock solution, which was dispensed into 200. Mu.L centrifuge tubes and stored at-80℃in the dark. Before use, the culture medium without serum is prepared into 200nmol/L working solution, and the working solution is stored at 4 ℃ in a dark place.
(3) MitosoxRed dye: the dye was dissolved in DMSO and 13. Mu.l DMSO was added to 50. Mu.g of the powder to prepare a 5mmol/L stock solution which was stored at-80℃in the absence of light. Before use, the culture medium without serum is prepared into 5 mu mol/L working solution, and the working solution is stored at 4 ℃ in a dark place.
(4) calceinAM dye: the calceinAM powder was dissolved in DMSO to prepare a stock solution with a final concentration of 1mmol/L and stored at-80℃in the dark. Once prepared, the stock solution should be used for a short period of time and should not be repeatedly freeze-thawed. Simultaneously, the working concentration of the CoCl2 stock solution is 1 mol/L. 0.5. Mu.L of AlceinAM stock solution and 1. Mu.L of LCoCl were added to 1mL of serum-free blank medium prior to use 2 The storage solution is stored at 4 ℃ in a dark place.
After the drug treatment, the supernatant was discarded, 100. Mu.L of each of the 5 kinds of prepared working solutions was added to each well, and the mixture was placed in an incubator and incubated for 1 hour in a dark place.
After the incubation was completed, the cell dye in the plates was discarded, and after washing the cells 1-2 times with 150. Mu.LPBS, the supernatant was discarded and 100. Mu.L of DMEM medium without serum was added.
And (5) immediately loading the image on a machine for image acquisition and analysis after the treatment is finished.
The high content collection mode collects 4 views for each well, and the collection conditions are as follows:
and (4) collecting 350nm/461nm wavelength to detect Hoechst33342 labeling and dyeing in the first pore channel.
And collecting 550nm/576nm wavelength detection TMRM, mitoSoxRed dye for dyeing.
And collecting 490nm/515nm wavelength to detect the calceinAM dye for dyeing.
And analyzing the obtained picture information by using a Harmony analysis system.
The number of nuclei is: and collecting the fluorescent number of the dye in the first pore canal for statistical analysis.
Mitochondrial membrane potential is: and collecting the average fluorescence intensity in the second channel for statistical analysis.
Mitochondrial active oxygen levels were: and collecting the average fluorescence intensity in the second channel for statistical analysis.
The mitochondrial mass is: and collecting the average fluorescence intensity in the second channel for statistical analysis.
Mitochondrial membrane permeability is: and collecting the average fluorescence intensity in the third channel for statistical analysis.
The results of the mitochondrial effect of bakuchiol on AML12 cells are shown in FIGS. 1-3, and the number of AML12 cells is significantly reduced after 24h of bakuchiol treatment. The TMRM fluorescence intensity of the dosing group is significantly reduced, indicating that bakuchiol reduces the mitochondrial membrane potential of AML12 cells. The fluorescence intensity of Mitosoxed and CalceinAM of the administration group is obviously increased, which suggests that the level of mitochondrial superoxide and mitochondrial membrane permeability of AML12 cells can be obviously improved after the treatment of bakuchiol. Comprehensive analysis shows that mitochondrial toxicity effect is the main cause of hepatotoxicity caused by fructus Psoraleae.
Example 2: high content screening system for analyzing mitochondrial toxicity effect of drug on myocardial cells
AC16 cells were collected and counted as described above and the cell concentration was adjusted to 10 4 Mu L/well is inoculated into a black 96-well plate and placed in an incubator for culture until the cell growth density reaches about 80%.
A blank control group and a doxorubicin administration group were set, and AC16 cells were treated with 1%o DMSO and a DMEM/F12 complete medium containing doxorubicin (5. Mu. Mol/L) for 24 hours, respectively, with 5 duplicate wells per group.
The preparation method of the high content dye comprises the following steps:
(1) nucleus dye Hoechst33342: hoechst33342 is added into DMSO to prepare a mother solution of 5mg/mL, the mother solution is split into 200 mu L centrifuge tubes and stored at-80 ℃ in a dark place. Before use, the working solution is prepared into 2 mug/mL by using a culture medium without serum, and the working solution is stored at 4 ℃ in a dark place.
(2) TMRM dye: the dye was dissolved in DMSO to prepare a 10mmol/L stock solution, which was dispensed into 200. Mu.L centrifuge tubes and stored at-80℃in the dark. Before use, the culture medium without serum is prepared into 200nmol/L working solution, and the working solution is stored at 4 ℃ in a dark place.
(3) MitosoxRed dye: the dye was dissolved in DMSO and 13. Mu.l DMSO was added to 50. Mu.g of the powder to prepare a 5mmol/L stock solution which was stored at-80℃in the absence of light. Before use, the culture medium without serum is prepared into 5 mu mol/L working solution, and the working solution is stored at 4 ℃ in a dark place.
(4) calceinAM dye: the calceinAM powder was dissolved in DMSO to prepare a stock solution with a final concentration of 1mmol/L and stored at-80℃in the dark. Once prepared, the stock solution should be used for a short period of time and should not be repeatedly freeze-thawed. Simultaneously, the working concentration of the CoCl2 stock solution is 1 mol/L. 0.5. Mu.L of AlceinAM stock solution and 1. Mu.L of LCoCl were added to 1mL of serum-free blank medium prior to use 2 The storage solution is stored at 4 ℃ in a dark place.
After the drug treatment, the supernatant was discarded, 100. Mu.L of each of the 5 kinds of prepared working solutions was added to each well, and the mixture was placed in an incubator and incubated for 1 hour in a dark place.
After the incubation was completed, the cell dye in the plates was discarded, and after washing the cells 1-2 times with 150. Mu.LPBS, the supernatant was discarded and 100. Mu.L of DMEM medium without serum was added.
And (5) immediately loading the image on a machine for image acquisition and analysis after the treatment is finished.
The high content collection mode collects 4 views for each well, and the collection conditions are as follows:
and (4) collecting 350nm/461nm wavelength to detect Hoechst33342 labeling and dyeing in the first pore channel.
And collecting 550nm/576nm wavelength detection TMRM, mitosoxRed dye for dyeing.
And collecting 490nm/515nm wavelength to detect the calceinAM dye for dyeing.
And analyzing the obtained picture information by using a Harmony analysis system.
The number of nuclei is: and collecting the fluorescent number of the dye in the first pore canal for statistical analysis.
Mitochondrial membrane potential is: and collecting the average fluorescence intensity in the second channel for statistical analysis.
Mitochondrial active oxygen levels were: and collecting the average fluorescence intensity in the second channel for statistical analysis.
Mitochondrial membrane permeability is: and collecting the average fluorescence intensity in the third channel for statistical analysis.
The effect of doxorubicin on AC16 cells at 5 μmol/L dose was shown in figures 4-6, and the cell number was significantly reduced 24h after doxorubicin treatment of AC16 cells. The administered group showed reduced TMRM fluorescence intensity, but no significant difference, indicating less effect of doxorubicin on mitochondrial membrane potential levels. The fluorescence intensity of Mitosoxed and CalceinAM of the administration group is obviously increased, which suggests that the mitochondrial superoxide level and mitochondrial membrane permeability of the myocardial cells can be obviously improved after doxorubicin treatment. Comprehensive analysis, doxorubicin exerts a toxic effect primarily by affecting cardiomyocyte mitochondrial oxidative stress levels and mitochondrial membrane permeability.
Example 3: the high content screening system compares the mitochondrial toxicity intensity of the multi-component fructus psoraleae
HepG2 cells were collected and counted as described above, and the cell concentration was adjusted to 10 4 Mu L/well is inoculated into a black 96-well plate and placed in an incubator for culture until the cell growth density reaches about 80%.
A blank control group, an isopsoralen administration group and a psoralen administration group were set, hepG2 cells were treated with DMEM complete medium containing 1% DMSO and, isopsoralen (15. Mu. Mol/L) and psoralen (15. Mu. Mol/L) for 24 hours, respectively, and 5 duplicate wells were set in each group.
The preparation method of the high content dye comprises the following steps:
(1) nucleus dye Hoechst33342: hoechst33342 is added into DMSO to prepare a mother solution of 5mg/mL, the mother solution is split into 200 mu L centrifuge tubes and stored at-80 ℃ in a dark place. Before use, the working solution is prepared into 2 mug/mL by using a culture medium without serum, and the working solution is stored at 4 ℃ in a dark place.
(2) TMRM dye: the dye was dissolved in DMSO to prepare a 10mmol/L stock solution, which was dispensed into 200. Mu.L centrifuge tubes and stored at-80℃in the dark. Before use, the culture medium without serum is prepared into 200nmol/L working solution, and the working solution is stored at 4 ℃ in a dark place.
(3) MitosoxRed dye: the dye was dissolved in DMSO and 13. Mu.l DMSO was added to 50. Mu.g of the powder to prepare a 5mmol/L stock solution which was stored at-80℃in the absence of light. Before use, the culture medium without serum is prepared into 5 mu mol/L working solution, and the working solution is stored at 4 ℃ in a dark place.
(4) calceinAM dye: dissolving calceinAM powder in DMSO,the stock solution was prepared to a final concentration of 1mmol/L and stored at-80℃in the dark. Once prepared, the stock solution should be used for a short period of time and should not be repeatedly freeze-thawed. Simultaneously, the working concentration of the CoCl2 stock solution is 1 mol/L. 0.5. Mu.L of AlceinAM stock solution and 1. Mu.L of LCoCl were added to 1mL of serum-free blank medium prior to use 2 The storage solution is stored at 4 ℃ in a dark place.
After the drug treatment, the supernatant was discarded, 100. Mu.L of each of the 5 kinds of prepared working solutions was added to each well, and the mixture was placed in an incubator and incubated for 1 hour in a dark place.
After the incubation was completed, the cell dye in the plates was discarded, and after washing the cells 1-2 times with 150. Mu.LPBS, the supernatant was discarded and 100. Mu.L of DMEM medium without serum was added.
And (5) immediately loading the image on a machine for image acquisition and analysis after the treatment is finished.
The high content collection mode collects 4 views for each well, and the collection conditions are as follows:
and (4) collecting 350nm/461nm wavelength to detect Hoechst33342 labeling and dyeing in the first pore channel.
And collecting 550nm/576nm wavelength detection TMRM, mitosoxRed dye for dyeing.
And collecting 490nm/515nm wavelength to detect the calceinAM dye for dyeing.
And analyzing the obtained picture information by using a Harmony analysis system.
The number of nuclei is: and collecting the fluorescent number of the dye in the first pore canal for statistical analysis.
Mitochondrial membrane potential is: and collecting the average fluorescence intensity in the second channel for statistical analysis.
Mitochondrial active oxygen levels were: and collecting the average fluorescence intensity in the second channel for statistical analysis.
The mitochondrial mass is: and collecting the average fluorescence intensity in the second channel for statistical analysis.
Mitochondrial membrane permeability is: and collecting the average fluorescence intensity in the third channel for statistical analysis.
Both psoralen and isopsoralen are active ingredients in psoralea. AML12 cells were treated with 15. Mu. Mol/L of the two drugs for 24h, and the results are shown in FIGS. 7-9. After 24 hours of treatment of the cell administration by isopsoralen, the mitochondrial membrane potential level of the AML12 cell is obviously reduced, the mitochondrial superoxide level and the mitochondrial membrane permeability are obviously increased, and the psoralen has no obvious influence on the mitochondrial membrane potential, the mitochondrial oxidative stress level and the mitochondrial membrane permeability of the AML12 cell. The results show that the mitochondrial toxicity damage of isopsoralen to AML12 cells is far higher than that of psoralen at the same concentration.
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.

Claims (9)

1. A drug mitochondrial toxicity detection method is characterized in that: the detection method uses a high content screening system as a technical means, and performs imaging analysis on living cells added with the drug to be detected based on a mitochondrial specific reaction fluorescent probe, so that the influence of the drug to be detected on the mitochondrial membrane potential, mitochondrial membrane permeability, mitochondrial active oxygen level and mitochondrial quality of the living cells is clear.
2. The method for detecting mitochondrial toxicity of a drug according to claim 1, wherein: the living cells used in the detection method are mouse normal liver cells AML12, human normal liver cells L02, human liver cancer cells HepG2, human renal tubular epithelial cells HK-2, human cardiac muscle cells AC16, rat cardiac muscle cells H9C2 and other cell types.
3. The method for detecting mitochondrial toxicity of a drug according to claim 1, wherein: in the detection method, the living cells need to be stained by using a dye before imaging analysis.
4. A method for detecting mitochondrial toxicity of a drug according to claim 3, wherein: the dye used for living cell nuclei is Hoechst33342 or DAPI.
5. A method for detecting mitochondrial toxicity of a drug according to claim 3, wherein: among the dyes, the dyes used for detecting mitochondrial membrane potential are tetramethyl rhodamine methyl ester, tetramethyl rhodamine ethyl ester, JC-1, rhodamine 123, mito-tracker red CMXRos, tetramethyl rhodamine methyl ester perchlorate or rhodamine 6G.
6. A method for detecting mitochondrial toxicity of a drug according to claim 3, wherein: the dye for detecting the mitochondrial membrane permeability is calcein AM or cobalt chloride composition.
7. A method for detecting mitochondrial toxicity of a drug according to claim 3, wherein: the dye for detecting mitochondrial active oxygen in the dye is Mitosox or MitopY1.
8. A method for detecting mitochondrial toxicity of a drug according to claim 3, wherein: among the dyes used for detecting mitochondrial mass are 10-nonylacridine orange bromide, mitoIDRed, mitotrackerGreen, mitotracker deep red633 or mitotracker red580.
9. An application of a drug mitochondrial toxicity detection method is characterized in that: the detection method can be used for screening mitochondrial toxic drugs, and can be used for safety evaluation and toxicology research of chemical drugs and traditional Chinese medicines.
CN202310922547.6A 2023-07-26 2023-07-26 Drug mitochondrial toxicity detection method and application thereof Pending CN116908158A (en)

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