JP6468781B2 - Method for evaluating mitochondrial membrane permeability, screening method for substances that suppress mitochondrial membrane permeability enhancement, screening method for substances that enhance mitochondrial membrane permeability, and inhibitor for enhancing mitochondrial membrane permeability - Google Patents

Description

  The present invention relates to a method for evaluating mitochondrial membrane permeability, a screening method for a substance that suppresses enhancement of mitochondrial membrane permeability, a screening method for a substance that enhances mitochondrial membrane permeability, and an inhibitor for enhancing mitochondrial membrane permeability.

  The loss of cranial neurons due to neuronal cell death or the like results in a decline in cognitive ability and motor sensory function. As this progresses, social life will eventually become difficult. Increasing cases of aging, Parkinson's disease, Alzheimer's disease, etc. is the biggest issue in Japan with aging. Currently, there is a demand for the development of neuroprotective therapies with drugs that prevent neuronal cell death and prevent or delay the onset of disease, some of which are being experimentally implemented.

  So far, the mechanism of nerve cell protective action has been clarified in cell and animal models of neurodegenerative diseases (Non-patent Documents 1 and 2). Oxidative stress, deficiency of neurotrophic factor, or neurotoxin activates the cell death mechanism of mitochondria, leading to cell death in the form of apoptosis. When a channel is formed and opened in the mitochondrial membrane, a protein that induces apoptosis flows into the cytoplasm, thereby causing cell death. Nerve cell protective drugs block each stage of cell death and ultimately protect nerve cells.

  In the process of cell death, an apoptotic signal is induced in mitochondria, activates caspase in the cytoplasm, causes nuclear DNA division and cell shrinkage, shows a typical apoptotic image, and leads to cell death. Measurement of each stage of this process includes measurement of changes in mitochondrial membrane permeation transition pore (PTP) formation, measurement of changes in mitochondrial membrane permeability based on measurement of changes in mitochondrial function, and outflowed apoptosis Measurement of inducing molecules, measurement of caspase activation, measurement of cell death based on quantification of apoptosis, and the like are known.

Maruyama W et al. (2001), J Neurochem, 78, pp. 727-735. Naoi M, et al. (2013), J Neural Transm, 120, pp. 1539-1551.

  As described above, although methods for measuring each stage of cell death are known, it has been difficult to measure and evaluate the first stage of cell death. For example, in measuring changes in mitochondrial membrane permeability, changes in membrane potential are detected by a fluorescence method, but changes in membrane potential are secondary changes. In addition, in measuring changes in mitochondrial function, in addition to the need for mitochondrial purification, it is difficult to evaluate the function of the entire cell.

  Accordingly, an object of the present invention is to provide a method for evaluating mitochondrial membrane permeability, which can evaluate the first stage of cell death. Another object of the present invention is to provide a screening method for a substance that suppresses enhancement of mitochondrial membrane permeability, a screening method for a substance that enhances mitochondrial membrane permeability, and an inhibitor for enhancing mitochondrial membrane permeability.

As described in detail in the examples of the present specification, the inventors of the present invention, in the process of inducing apoptosis of neurons, increase the mitochondrial membrane permeability and increase the amount of Ca ²⁺ in the cytoplasm and in the reaction solution (extracellular). It was found that an increase in the amount of superoxide (O ₂ ⁻ ) was observed, and that the increase in the amount of Ca ²⁺ and O ₂ ⁻ was the initial signal for inducing apoptosis. The present invention is based on these new findings.

That is, the present invention relates to a method for evaluating mitochondrial membrane permeability, comprising adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing test cells in vitro, and using the calcium ion indicator as an index, And a step of comparing the measured amount of calcium ions with a first reference value, and when the measured amount of calcium ions exceeds the first reference value, the mitochondria of the test cell The present invention relates to an evaluation method in which membrane permeability is evaluated to be enhanced.

According to the said evaluation method, since evaluation based on the amount of calcium ions is performed, a change in mitochondrial membrane permeability can be rapidly evaluated without requiring complicated pretreatment. Therefore, the evaluation method can evaluate the first stage of cell death. Conventionally, the involvement of Ca ²⁺ in cell death has been examined as an increase / decrease in Ca ²⁺ inside and outside the cell and disturbance of homeostasis. In contrast, in the present invention, a cell death signal is measured as the kinetics of Ca ²⁺ in mitochondria.

  The evaluation method includes a step of adding an active oxygen indicator to the reaction solution in vitro, a step of measuring an active oxygen amount using the active oxygen indicator as an index, a measured active oxygen amount, and a second reference value, Preferably further comprising the step of comparing In this case, when the measured calcium ion amount exceeds the first reference value and the measured active oxygen amount exceeds the second reference value, the mitochondrial membrane permeability of the test cell is enhanced. It is evaluated.

  By performing evaluation based on the amount of active oxygen in addition to the amount of calcium ions, changes in mitochondrial membrane permeability related to cell death can be evaluated more accurately.

The active oxygen may be superoxide (O ₂ ⁻ ). Thereby, the change in mitochondrial membrane permeability associated with cell death can be evaluated with higher accuracy.

  In the evaluation method, preferably, at least one of the calcium ion indicator and the active oxygen indicator is a fluorescent indicator, the calcium ion indicator is a fluorescent indicator, and the active oxygen indicator is a chemiluminescent indicator. preferable. Thereby, the amount of calcium ions and the amount of active oxygen can be easily detected, and the detection sensitivity can be improved.

  In the above evaluation method, since the evaluation is performed based on the calcium ion amount or the active oxygen amount, an initial signal for inducing apoptosis due to increased permeability of the mitochondrial membrane can be detected. Therefore, in the evaluation method, the enhancement of mitochondrial membrane permeability of the test cell may indicate induction of apoptosis. That is, when the measured calcium ion amount exceeds the first reference value and / or when the measured active oxygen amount exceeds the second reference value, the test cell is induced to be apoptotic. Be evaluated.

The present invention also provides a method for screening a substance that suppresses increased mitochondrial membrane permeability, comprising adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing a plurality of test cells in vitro, and the reaction solution comprising: Dividing into at least two and obtaining a reaction liquid containing the first test cell and a reaction liquid containing the second test cell, the test substance and the mitochondrial membrane permeability enhancer are brought into contact with the first test cell. , Contacting the second test cell with a mitochondrial membrane permeability enhancer, and measuring the amount of calcium ions for each of the first test cell and the second test cell using the calcium ion indicator as an index Comparing the amount of calcium ions measured for the first test cell and the second test cell And when the amount of calcium ions measured for the first test cell is lower than the amount of calcium ions measured for the second test cell, the test substance is a substance that suppresses the enhancement of mitochondrial membrane permeability. It also relates to screening methods that are evaluated as being.

  In the above screening method, mitochondrial membrane permeability can be evaluated because the evaluation is based on the amount of calcium ions. Therefore, if the amount of calcium ions is reduced when both the mitochondrial membrane permeability enhancer and the test substance are administered compared to the case where the mitochondrial membrane permeability enhancer alone is administered, the test substance is permeated through the mitochondrial membrane. It can be evaluated as a substance that suppresses sex enhancement. In addition, since the screening method performs evaluation based on the amount of calcium ions, complicated pretreatment is not required, and changes in mitochondrial membrane permeability can be detected quickly, so that evaluation in a short time is possible.

  The screening method includes a step of adding an active oxygen indicator to a reaction solution containing the plurality of test cells in vitro, and the first test cell and the second test cell, respectively, using the active oxygen indicator as an index. Preferably, the method further includes the step of measuring the amount of active oxygen and the step of comparing the amount of active oxygen measured for the first test cell and the second test cell. In this case, the amount of calcium ions measured for the first test cell is less than the amount of calcium ions measured for the second test cell, and the amount of active oxygen measured for the first test cell is When the amount of active oxygen measured for 2 test cells is below, the test substance is evaluated as a substance that suppresses the enhancement of mitochondrial membrane permeability.

  By performing the evaluation based on the amount of active oxygen in addition to the amount of calcium ions, the evaluation can be made more accurately.

The active oxygen may be superoxide (O ₂ ⁻ ).

  In the screening method, preferably, at least one of the calcium ion indicator and the active oxygen indicator is a fluorescent indicator, the calcium ion indicator is a fluorescent indicator, and the active oxygen indicator is a chemiluminescent indicator. preferable. Thereby, the amount of calcium ions and the amount of active oxygen can be easily detected, and the detection sensitivity can be improved.

  In the above screening method, since the evaluation is performed based on the calcium ion amount or the active oxygen amount, an initial signal for inducing apoptosis due to increased permeability of the mitochondrial membrane can be detected. Therefore, in the screening method, the enhancement of mitochondrial membrane permeability of the test cell may indicate induction of apoptosis. That is, the screening method may evaluate that the test substance is a substance that suppresses the induction of apoptosis.

  The present invention further relates to an inhibitor for enhancing mitochondrial membrane permeability, which contains sesamin or sesaminol as an active ingredient.

  The present inventors have found that sesamin and sesaminol are substances that suppress the enhancement of mitochondrial membrane permeability. The enhancement inhibitor is based on this novel finding.

  Since the above-mentioned enhancement inhibitor can suppress enhancement of mitochondrial membrane permeability in the process of inducing apoptosis of nerve cells, it can also be suitably used as an apoptosis inhibitor.

The present invention is also a method for screening a substance that enhances mitochondrial membrane permeability, comprising adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing a plurality of test cells in vitro, and at least two reaction solutions. The reaction solution containing the first test cell and the reaction solution containing the second test cell are obtained, the test substance is brought into contact with the first test cell, and the second test cell is tested. A step of not contacting a substance, a step of measuring the amount of calcium ions for each of the first test cell and the second test cell, using the calcium ion indicator as an index, the first test cell, and the above Comparing the amount of calcium ion measured for the second test cell, and measuring calcium for the first test cell If the ion amount exceeds the amount of calcium ions was measured for the second test cell, the test substance is evaluated as a substance that enhances the mitochondrial membrane permeability, also relates to a screening method.

  In the above screening method, mitochondrial membrane permeability can be evaluated because the evaluation is based on the amount of calcium ions. Therefore, if the amount of calcium ions is increased when the test substance is contacted compared to the case where the test substance is not contacted, the test substance can be evaluated as a substance that enhances mitochondrial membrane permeability. . In addition, since the screening method performs evaluation based on the amount of calcium ions, complicated pretreatment is not required, and changes in mitochondrial membrane permeability can be detected quickly, so that evaluation in a short time is possible.

  The screening method includes a step of adding an active oxygen indicator to a reaction solution containing the plurality of test cells in vitro, and the first test cell and the second test cell, respectively, using the active oxygen indicator as an index. Preferably, the method further includes the step of measuring the amount of active oxygen and the step of comparing the amount of active oxygen measured for the first test cell and the second test cell. In this case, the amount of calcium ions measured for the first test cell exceeds the amount of calcium ions measured for the second test cell, and the amount of active oxygen measured for the first test cell is When the amount of active oxygen measured for the two test cells is exceeded, the test substance is evaluated as a substance that enhances mitochondrial membrane permeability.

  By performing the evaluation based on the amount of active oxygen in addition to the amount of calcium ions, the evaluation can be made more accurately.

The active oxygen may be superoxide (O ₂ ⁻ ).

  In the screening method, preferably, at least one of the calcium ion indicator and the active oxygen indicator is a fluorescent indicator, the calcium ion indicator is a fluorescent indicator, and the active oxygen indicator is a chemiluminescent indicator. preferable. Thereby, the amount of calcium ions and the amount of active oxygen can be easily detected, and the detection sensitivity can be improved.

  In the above screening method, since the evaluation is performed based on the calcium ion amount or the active oxygen amount, an initial signal for inducing apoptosis due to increased permeability of the mitochondrial membrane can be detected. Therefore, in the screening method, the enhancement of mitochondrial membrane permeability of the test cell may indicate induction of apoptosis. That is, the screening method may evaluate that the test substance is a substance that induces apoptosis.

  ADVANTAGE OF THE INVENTION According to this invention, the mitochondrial membrane permeability evaluation method which can evaluate the initial stage of cell death is provided. According to the evaluation method of the present invention, it is possible to evaluate the first stage of the cell death signal activity, which is difficult with the conventional method, without performing complicated pretreatment.

  In addition, according to the present invention, a screening method for a substance that suppresses enhancement of mitochondrial membrane permeability is provided. The screening method of the present invention does not require complicated pretreatment, and can evaluate a test substance that can suppress the enhancement of mitochondrial membrane permeability related to cell death in a short time. Therefore, for example, the apoptosis induction inhibitory activity, nerve cell protective activity, etc. of natural compounds or synthetic compounds derived from foods can be measured and evaluated quickly and easily.

  In Japan, which has entered a super-aged society for the first time in the world, high interest is expected in the prevention of cranial neurodegenerative diseases, which are a major cause of hindering healthy longevity. In addition, as the expectation for preventive medicine by “food” is increasing, a method that can quickly and easily measure apoptosis-inducing inhibitory activity and nerve cell protective activity for substances that can be applied to food is a realization of a healthy and long-lived society. It is thought that it can contribute greatly.

^{Ca 2+} amounts in the cytoplasm due to the addition of the mitochondrial membrane permeability enhancers (PK11195), and the reaction solution in the _{O 2} ^- is a graph showing changes in amount. ^{Ca 2+} amounts in the cytoplasm due to the addition of the mitochondrial membrane permeability enhancers (PK11195) and neuroprotective agents (cyclosporin A), and the reaction solution in the _{O 2} ^- is a graph showing changes in amount. It is a graph which shows the change of the amount of Ca < ²⁺ > in cytoplasm by addition of a mitochondrial membrane permeation enhancer (PK11195) and a neurocyte protective agent (Rasagiline). Ca ²⁺ amounts in the cytoplasm due to the addition of the food ingredients (sesamin and sesaminol), and the reaction solution in the O ₂ ^- is a graph showing changes in amount. It is a schematic cross section of the simultaneous measurement apparatus of fluorescence and chemiluminescence.

  Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[Evaluation method of mitochondrial membrane permeability]
The method for evaluating mitochondrial membrane permeability according to this embodiment includes a step of adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing test cells in vitro, and a step of measuring the amount of calcium ions using the calcium ion indicator as an index. And comparing the measured amount of calcium ions with a first reference value.

  As a test cell, a cell type whose mitochondrial membrane permeability should be evaluated may be used, and any cell can be used. Examples of test cells include nerve cells, glial cells, cardiomyocytes, cancer cells, hepatocytes, and cultured cell lines that are systematized from these cells.

  The reaction solution containing the test cell can be prepared by suspending the test cell in a buffer solution (for example, RH buffer solution, PBS (−) buffer solution) or the like.

  The calcium ion indicator may be any reagent that can detect a signal correlated with the abundance of calcium ions. For example, a fluorescent indicator that reacts quantitatively with calcium ions, a luminescent (eg, chemiluminescent) indicator, or an absorption indicator. Can be used. A commercially available calcium ion indicator may be used. As such an indicator, for example, Fluo-3 (manufactured by DOJINDO), Indo-1, Indo-5F, bis-fura-2, Quin-2, Quin-AM, Rhod-2, X-Rhod-1 (any) Also manufactured by Molecular Probes).

  Examples of the method of adding a calcium ion indicator to a reaction solution containing test cells include a method of adding the above-described calcium ion indicator or a solution thereof to the reaction solution. The calcium ion indicator added to the reaction solution reacts with calcium ions in the reaction solution that permeates the mitochondrial membrane and the cell membrane, or is taken into the cell of the test cell by the cell membrane permeability of the calcium ion indicator itself, and the mitochondrial membrane It reacts with the calcium ions in the cytoplasm that have permeated through.

Since the step of adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing test cells in vitro can more quickly evaluate changes in mitochondrial membrane permeability, calcium ions (Ca ²⁺ ) enter the cells of test cells in vitro. The step of adding an indicator is preferred.

  Examples of a method for adding a calcium ion indicator into the cells of a test cell include, for example, a method in which a calcium ion indicator is added to a reaction solution using the cell membrane permeability of the calcium ion indicator itself to move into the cell, A method of introducing an indicator and a reagent commonly used for introducing a substance into the cell (for example, lipofection reagent) and adding it to the reaction solution, and then physically introducing the calcium ion indicator into the cell Examples thereof include a method (for example, a microinjection method and an electroporation method).

  Measurement of the amount of calcium ions in the reaction solution or in the cytoplasm is performed by a conventional method using a calcium ion indicator as an index. For example, when the calcium ion indicator is a fluorescent indicator, the amount of calcium ions is measured by irradiating a sample container containing a test cell with excitation light output from an excitation light source and detecting the emitted fluorescence with a fluorescence detector. be able to. When the calcium ion indicator is a chemiluminescence indicator, the amount of calcium ions can be measured by detecting the chemiluminescence released from the sample container containing the test cell with a chemiluminescence detector. Since changes in mitochondrial membrane permeability can be evaluated more accurately and more quickly, it is preferable to measure the amount of calcium ions in the cytoplasm.

  Subsequently, the measured calcium ion amount is compared with the first reference value. The first reference value can be set as needed according to the purpose of evaluation, the cell type to be evaluated, and the degree of mitochondrial membrane permeability as a reference. More specifically, the amount of calcium ions measured for a cell in a state serving as a comparative control can be set as the first reference value. For example, when the purpose is to detect the initial signal of apoptosis induction in nerve cells, the cells are cultured under conditions that do not induce apoptosis (for example, conditions without stress such as oxidative stress, neurotrophic factor deficiency, or neurotoxin). It is possible to measure the amount of calcium ions in the nerve cells, and set the measured amount of calcium ions as the first reference value.

  When the measured amount of calcium ions exceeds the first reference value, the mitochondrial membrane permeability of the test cell is enhanced compared to the cell in the state of comparison used to set the first reference value It is evaluated.

  The mitochondrial membrane permeability evaluation method of the present embodiment may perform evaluation based on the amount of active oxygen as well as evaluation based on the amount of calcium ions. That is, the method for evaluating mitochondrial membrane permeability of the present embodiment includes a step of adding an active oxygen indicator to a reaction solution containing test cells in vitro, a step of measuring the amount of active oxygen using the active oxygen indicator as an index, and a measurement The method may further include a step of comparing the determined amount of active oxygen with the second reference value. Thereby, the change of the mitochondrial membrane permeability relevant to cell death can be evaluated more accurately.

The active oxygen indicator may be any reagent that can detect a signal that correlates with the amount of active oxygen present. For example, a fluorescent indicator that reacts quantitatively with active oxygen, a luminescent (eg, chemiluminescent) indicator, or an absorption indicator. Can be used. A commercially available active oxygen indicator may be used. Examples of such indicators include 2-methyl-6-phenyl-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (CLA), 2-methyl-6- (4-methoxyphenyl) -3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA), 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone (MPEC), indocyanine type imidazopyranodine compound ( NIR-CLA), 2- [2,4,5,7-tetrafluoro-6- (2-nitro-4,5-dimethoxyphenylsulfonyloxy) -3-oxo-3H-xanthen-9-yl] benzoic acid (BES-So) Indicators that react with superoxide such as dihydroethidium, carboxy-H ₂ DCFDA (2 ′, 7′-dichlorodihydrofluorescein di Acetate), dihydrocalcein, dihydrorhodamine 123, indicators that react with hydrogen peroxide such as luminol, CM-H ₂ DCFDC, proxylfluorescamine, indicators that react with hydroxy radicals such as TEPO-9-AC, trans-1 Examples include indicators that react with singlet oxygen such as-(2'-methoxyvinyl) pyrene.

  Examples of a method for adding an active oxygen indicator to a reaction solution containing test cells include a method of adding the above-described active oxygen indicator or a solution thereof to the reaction solution. The active oxygen indicator added to the reaction solution reacts with the active oxygen in the reaction solution, or is taken into the cell of the test cell by the cell membrane permeability of the active oxygen indicator itself and reacts with the active oxygen in the cytoplasm. become.

  The step of adding the active oxygen indicator to the reaction solution containing the test cell in vitro is a step of adding the active oxygen indicator into the cell of the test cell in vitro since the change in mitochondrial membrane permeability can be evaluated more quickly. Also good.

  Examples of the method for adding an active oxygen indicator into the cells of a test cell include the same method as the method for adding a calcium ion indicator. When the active oxygen indicator is added, the order of adding the active oxygen indicator and the calcium ion indicator is arbitrary, and they may be added simultaneously.

  Measurement of the amount of active oxygen in the reaction solution or in the cytoplasm is performed by a conventional method using an active oxygen indicator as an index. Specific examples of the method for measuring the amount of active oxygen include the same methods as the specific examples of the method for measuring the amount of calcium ions. Since changes in mitochondrial membrane permeability can be evaluated with higher accuracy, it is preferable to measure the amount of active oxygen in the reaction solution.

  In the measurement of the amount of calcium ions and the amount of active oxygen, from the viewpoint of improving detection sensitivity, at least one of the calcium ion indicator and the active oxygen indicator is preferably a fluorescent indicator, the calcium ion indicator is a fluorescent indicator, and More preferably, the active oxygen indicator is a chemiluminescent indicator.

  The amount of calcium ions and the amount of active oxygen may be measured substantially simultaneously. Thereby, the amount of calcium ions and the amount of active oxygen under the same conditions can be measured, and mitochondrial membrane permeability can be more accurately evaluated.

  The amount of calcium ions and the amount of active oxygen can be measured using, for example, a fluorescence detection device, a chemiluminescence detection device, or an absorbance measurement device. When the amount of calcium ions and the amount of active oxygen are measured substantially simultaneously by fluorescence and chemiluminescence, for example, the fluorescence and chemiluminescence described in JP-A-2004-61438, JP-A-2000-13234, etc. are simultaneously measured. It can also be measured using an apparatus.

  Subsequently, the measured amount of active oxygen is compared with the second reference value. The second reference value can be set at any time according to the evaluation purpose, the cell type to be evaluated, and the degree of mitochondrial membrane permeability as a reference. More specifically, the amount of active oxygen measured for a cell in a state as a comparison control can be set as the second reference value. For example, when the purpose is to detect the initial signal of apoptosis induction in nerve cells, the cells are cultured under conditions that do not induce apoptosis (for example, conditions without stress such as oxidative stress, neurotrophic factor deficiency, or neurotoxin). It is possible to measure the amount of active oxygen in the nerve cells, and set the measured amount of active oxygen as the second reference value. The second reference value is preferably set under the same conditions as the first reference value.

  Examples of active oxygen include superoxide, hydrogen peroxide, hydroxy radical, and singlet oxygen. For the purpose of detecting the initial signal for inducing apoptosis, it is preferable to employ superoxide as the active oxygen because it enables more accurate evaluation.

  When the measured amount of active oxygen exceeds the second reference value, the mitochondrial membrane permeability of the test cell was enhanced compared to the cell in the state of comparison used to set the second reference value It is evaluated. Here, from the viewpoint of further improving the accuracy of evaluation, when the measured calcium ion amount exceeds the first reference value and the measured active oxygen amount exceeds the second reference value, the mitochondria of the test cell It is preferable to evaluate that the membrane permeability is enhanced.

According to the method for evaluating mitochondrial membrane permeability described above, an initial signal of apoptosis induction (for example, increase in Ca ²⁺ or O ₂ ⁻ amount in the cytoplasm) due to enhanced mitochondrial membrane permeability can be detected. The form evaluation method is also useful as an evaluation method or detection method of apoptosis induction in a test cell.

[Screening method for substances that suppress the increase in mitochondrial permeability]
The present invention can also be applied to a screening method for substances that suppress the enhancement of mitochondrial membrane permeability.

The method for screening a substance that suppresses the enhancement of mitochondrial membrane permeability according to this embodiment includes a step of adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing a plurality of test cells in vitro, and at least two reaction solutions. Separately, a reaction liquid containing the first test cell and a reaction liquid containing the second test cell are obtained, the test substance and the mitochondrial membrane permeation enhancer are brought into contact with the first test cell, and the second test A step of contacting a cell with a mitochondrial membrane permeability enhancer, a step of measuring the amount of calcium ions for each of the first test cell and the second test cell using the calcium ion indicator as an index, and the first test cell And comparing the amount of calcium ions measured for the second test cell. As a result of the comparison, if the amount of calcium ions measured for the first test cell is less than the amount of calcium ions measured for the second test cell, the test substance is evaluated as a substance that suppresses enhancement of mitochondrial membrane permeability Is done.

  There is no particular limitation on the test substance, and any substance can be used. Examples of the test substance include naturally occurring compounds, artificially synthesized compounds, peptides, proteins, lipids, and nucleic acids (ribonucleic acid, deoxyribonucleic acid, etc.). A food-derived substance may be used as the test substance.

  As the mitochondrial membrane permeability enhancer, a substance having the property of enhancing the permeability of the mitochondrial membrane can be used without limitation. The property of enhancing the permeability of the mitochondrial membrane is preferably the property of promoting PTP formation (opening) of the mitochondrial membrane. Specific examples of the mitochondrial permeability enhancer include PK11195 (1- (2-chlorophenyl) -N-methyl-N- (1-methylpropyl) -3-isoquinolinecarboxamide). PK11195 is known to cause cell death by opening PTP of the mitochondrial membrane and activating an apoptotic signal (Non-patent Document 2).

  Any cell may be used as the test cell. In particular, for the purpose of screening a substance having neuronal cell protective activity, it is preferable to use a neuronal cell or a cultured cell line systematized from neuronal cells, and SH-SY5Y cell which is a cultured cell line of dopamine cell line Is more preferable.

  In the screening method according to the present embodiment, a calcium ion indicator is first added to a reaction solution containing a plurality of test cells. The addition is performed in vitro. Specific examples of the calcium ion indicator and the method of adding the same are as described above.

  Next, the reaction solution is divided into at least two to obtain a reaction solution containing the first test cell and a reaction solution containing the second test cell. Then, the test substance and the mitochondrial membrane permeability enhancer are brought into contact with the first test cell, and the mitochondrial membrane permeability enhancer is brought into contact with the second test cell.

  As a method of bringing the test substance and the mitochondrial membrane permeability enhancer into contact with the test cell, for example, it can be performed by adding the test substance or the mitochondrial membrane permeability enhancer or a solution thereof to a reaction solution containing the test cell. it can. In the first test cell, the order in which the test substance and the mitochondrial membrane permeation enhancer are brought into contact with each other is arbitrary, and may be contacted at the same time. From the viewpoint of more accurately evaluating the effect of suppressing the enhancement of mitochondrial membrane permeability, it is preferable to contact a test substance with a test cell and then contact a mitochondrial membrane permeability enhancer.

  Next, using the calcium ion indicator as an index, the amount of calcium ions in the reaction solution or in the cytoplasm is measured for each of the first test cell and the second test cell. Specific examples of the measurement method are as described above.

  Next, the calcium ion content measured about the 1st test cell and the 2nd test cell is compared. In the second test cell, the permeability of the mitochondrial membrane is enhanced by the action of the mitochondrial membrane permeability enhancer, and the amount of calcium ions in the reaction solution or in the cytoplasm increases. On the other hand, when the test substance has the property of suppressing the enhancement of mitochondrial membrane permeability, the action of the mitochondrial membrane permeability enhancer is canceled by the action of the test substance in the first test cell, and the second test cell and In comparison, an increase in the amount of calcium ions in the reaction solution or in the cytoplasm is suppressed or does not increase. Therefore, when the amount of calcium ions measured for the first test cell is lower than the amount of calcium ions measured for the second test cell, the test substance is evaluated as a substance that suppresses the enhancement of mitochondrial membrane permeability. .

  In the screening method according to the present embodiment, a reaction solution containing a plurality of test cells to which a calcium ion indicator is added is divided into three, a reaction solution containing a first test cell, and a reaction solution containing a second test cell. And a reaction solution containing a third test cell, contacting the first test cell with a test substance and a mitochondrial membrane permeability enhancer, contacting the second test cell with a mitochondrial membrane permeability enhancer, It may include a step of contacting the third test cell with a cytoprotective drug and a mitochondrial membrane permeability enhancer. In addition, when the cytoprotective agent is a cytoprotective factor that functions as a cytoprotective agent when expressed in the cell like the Bcl-2 protein described later, the contact with the cytoprotective factor in the contacting step Means that the cytoprotective factor is expressed.

  As the cytoprotective agent (also referred to as cytoprotective agent) to be contacted with the third test cell, a substance having a property of suppressing the enhancement of mitochondrial membrane permeability can be used without limitation. Examples of cytoprotective agents include cyclosporin A (Cyclosporin-A), rasagiline (N-2-propynyl-1-indanamine), and Bcl-2 protein. Cyclosporine A is known to inhibit PTP opening. Bcl-2 protein and rasagiline are known to inhibit apoptosis by inhibiting PTP formation and protect neurons (Non-patent Document 1). The Bcl-2 protein may be a gene that is introduced into a test cell and expressed in mitochondria.

  In the third test cell, since the action of the mitochondrial membrane permeability enhancer is counteracted by the action of the cytoprotective agent, an increase in the amount of calcium ions in the reaction solution or in the cytoplasm is suppressed or does not increase. Therefore, in addition to comparing the calcium ion content measured for the first test cell and the second test cell, by comparing the calcium ion content measured for the first test cell and the third test cell. Therefore, more accurate evaluation can be performed.

  The screening method according to the present embodiment may perform evaluation based on the amount of active oxygen as well as evaluation based on the amount of calcium ions. That is, the screening method according to the present embodiment includes a step of adding an active oxygen indicator to the reaction solution containing the plurality of test cells in vitro, the first test cell, and the second test using the active oxygen indicator as an index. A step of measuring the amount of active oxygen for each of the cells and, if necessary, the third test cell, the first test cell, the second test cell, and, if necessary, the third test cell And comparing the amount of active oxygen. Thereby, the effect | action which suppresses the enhancement of mitochondrial membrane permeability relevant to cell death can be evaluated more accurately.

  Specific examples of the active oxygen indicator and the method of adding it are as described above. Moreover, the specific example of the measuring method of the amount of calcium ions and the amount of active oxygen is as above-mentioned.

  When the amount of active oxygen measured for the first test cell is lower than the amount of active oxygen measured for the second test cell, the test substance is evaluated as a substance that suppresses enhancement of mitochondrial membrane permeability. Here, from the viewpoint of further improving the accuracy of evaluation, the amount of calcium ions measured for the first test cell was lower than the amount of calcium ions measured for the second test cell, and was measured for the first test cell. When the amount of active oxygen is lower than the amount of active oxygen measured for the second test cell, it is preferable that the test substance is evaluated as a substance that suppresses enhancement of mitochondrial membrane permeability.

According to the screening method of the present embodiment, evaluation based on an initial signal of apoptosis induction due to enhanced mitochondrial membrane permeability (for example, an increase in the amount of Ca ²⁺ or O ₂ ⁻ in the cytoplasm) is possible. The screening method is also useful as a screening method for substances that suppress apoptosis.

  A test substance evaluated as a substance that suppresses enhancement of mitochondrial membrane permeability by the screening method of the present embodiment is suitably used as an agent for inhibiting mitochondrial membrane permeability enhancement. Next, the mitochondrial permeability enhancement inhibitor will be described.

[Inhibitors of enhanced mitochondrial permeability]
The mitochondrial permeability enhancement inhibitor of this embodiment contains sesamin or sesaminol as an active ingredient.

  Sesamin is one of the components contained in sesame, and is 5,5 ′-(1S, 3aR, 4S, 6aR) -tetrahydro-1H, 3H-furo [3,4-c] furan-1,4-diylbis. It is a compound also called (1,3-benzodioxole).

  Sesaminol is one of the components contained in sesame and is (1S, 3aβ, 6aβ) -1β- (6-hydroxy-1,3-benzodioxol-5-yl) -4β- (1,3 -Benzodioxol-5-yl) tetrahydro-1H, 3H-furo [3,4-c] furan.

  Since sesamin and sesaminol can suppress the increase in mitochondrial permeability induced by the mitochondrial permeability enhancer (PK11195), the enhancement of mitochondrial permeability that contains sesamin and sesaminol as active ingredients The inhibitor is suitably used for the purpose of suppressing the enhancement of mitochondrial membrane permeability.

  Sesamin and sesaminol may be naturally derived or chemically synthesized. Sesamin and sesaminol can be obtained by refining from sesame oil, for example. Commercially available sesamin and sesaminol may be used.

  The mitochondrial membrane permeability inhibitor of this embodiment may contain sesamin or sesaminol alone as an active ingredient, or a combination of two kinds as an active ingredient.

  The inhibitor for enhancing mitochondrial membrane permeability of the present embodiment may be any shape such as a solid, a liquid (including a suspension), and a paste. Further, the dosage form may be any of powder, pill, granule, tablet, syrup, troche, capsule and the like.

  The above-mentioned various preparations may be composed of only the active ingredient sesamin or sesaminol, or may be a mixture of the active ingredient and a pharmaceutically acceptable additive. Moreover, the above-mentioned formulation can be prepared by, for example, mixing sesamin, sesaminol, and, if necessary, an additive and forming the dosage form. When an additive is included, the content of the active ingredient is, for example, 0.1 to 90% by mass based on the total amount of the preparation.

  Examples of the pharmaceutically acceptable additive include an excipient, a binder, a lubricant, a disintegrant, an emulsifier, a surfactant, and a base.

  Examples of the excipient include lactose, sucrose, starch, dextrin and the like. Examples of the binder include polyvinyl alcohol, gum arabic, tragacanth, gelatin, hydroxypropylmethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc and the like. Examples of the disintegrant include crystalline cellulose, agar, gelatin, calcium carbonate, sodium bicarbonate, dextrin and the like. Examples of the emulsifier or surfactant include Tween 60, Tween 80, Span 80, and glyceryl monostearate. Examples of the base include cetostearyl alcohol, lanolin, polyethylene glycol, rice bran oil, fish oil (DHA, EPA, etc.), olive oil and the like.

  The mitochondrial permeability enhancement inhibitor of this embodiment may be administered to humans or non-human mammals. The dosage and administration method can be appropriately determined according to the condition, age, etc. of the individual to be administered. Suitable administration methods include, for example, oral administration. As an example of the dose and administration method, taking into account the active ingredient concentration of the mitochondrial membrane permeability enhancement inhibitor of this embodiment and the efficiency of translocation into the brain, 100 mg to 1 g (based on the active ingredient amount) is orally administered once a day. The method of administration can be mentioned.

  Mitochondrial membrane permeability enhancement inhibitor of this embodiment is a pharmaceutical, quasi-drug, general food, supplement, food for specified health use, functional food, dietary supplement, food additive, feed, feed additive, etc. Can be used.

  Since the inhibitor for enhancing mitochondrial membrane permeability of the present embodiment can suppress the enhancement of mitochondrial membrane permeability in the process of inducing apoptosis of cells, for example, apoptosis inhibitor, cytoprotective agent (for example, protection of nerve cells) As an agent). In addition, an anti-aging effect, an effect of keeping the brain young, and the like can also be achieved through an apoptosis-inhibiting action and a nerve cell protecting action.

[Screening method for substances that enhance mitochondrial membrane permeability]
The present invention can also be applied to screening methods for substances that enhance mitochondrial membrane permeability.

The method for screening a substance that enhances mitochondrial membrane permeability according to this embodiment comprises a step of adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing a plurality of test cells in vitro, and dividing the reaction solution into at least two. Obtaining a reaction solution containing the first test cell and a reaction solution containing the second test cell, contacting the test substance with the first test cell, and not contacting the test substance with the second test cell; The step of measuring the amount of calcium ions for each of the first test cell and the second test cell using the calcium ion indicator as an index, and the calcium ion measured for the first test cell and the second test cell Comparing the amounts. As a result of the comparison, when the amount of calcium ions measured for the first test cell exceeds the amount of calcium ions measured for the second test cell, the test substance is evaluated as a substance that enhances mitochondrial membrane permeability. .

  There is no particular limitation on the test substance, and any substance can be used. Examples of the test substance include naturally occurring compounds, artificially synthesized compounds, peptides, proteins, lipids, and nucleic acids (ribonucleic acid, deoxyribonucleic acid, etc.). A food-derived substance may be used as the test substance.

  Any cell may be used as the test cell. In particular, for the purpose of screening for substances having a cancer therapeutic effect, it is preferable to use cancer cells or cultured cell lines systematized from cancer cells. Since cancer cells have the property of continuing to proliferate while avoiding apoptosis, there are examples in which a substance that induces apoptosis is used as an anticancer agent or an anticancer adjuvant.

  In the screening method according to the present embodiment, a calcium ion indicator is first added to a reaction solution containing a plurality of test cells. The addition is performed in vitro. Specific examples of the calcium ion indicator and the method of adding the same are as described above.

  Next, the reaction solution is divided into at least two to obtain a reaction solution containing the first test cell and a reaction solution containing the second test cell. Then, the test substance is brought into contact with the first test cell, and the test substance is not brought into contact with the second test cell.

  As a method of bringing a test substance into contact with a test cell, for example, it can be performed by adding the test substance or a solution thereof to a reaction solution containing the test cell.

  Next, using the calcium ion indicator as an index, the amount of calcium ions in the reaction solution or in the cytoplasm is measured for each of the first test cell and the second test cell. Specific examples of the measurement method are as described above.

  Next, the calcium ion content measured about the 1st test cell and the 2nd test cell is compared. The second test cell is a control indicating the amount of calcium ions in the reaction solution or in the cytoplasm when not in contact with the test substance. When the test substance has the property of enhancing mitochondrial membrane permeability, the amount of calcium ions in the reaction solution or in the cytoplasm is higher in the first test cell than in the second test cell as a control due to the action of the test substance. Will rise. Therefore, when the amount of calcium ions measured for the first test cell exceeds the amount of calcium ions measured for the second test cell, the test substance is evaluated as a substance that enhances mitochondrial membrane permeability.

  In the screening method according to the present embodiment, the test substance and cytoprotective drug may be contacted with the first test cell, and the cytoprotective drug may be contacted with the second test cell. The cytoprotective drug is as described above. In the second test cell, the increase in permeability of the mitochondrial membrane is suppressed by the action of the cytoprotective drug. On the other hand, when the test substance has the property of enhancing mitochondrial membrane permeability, the first test cell is counteracted by the action of the test substance and the reaction of the first test cell compared to the second test cell. The amount of calcium ions in the fluid or in the cytoplasm will increase. Therefore, when the amount of calcium ions measured for the first test cell exceeds the amount of calcium ions measured for the second test cell, the test substance is evaluated as a substance that enhances mitochondrial membrane permeability.

  In the screening method according to the present embodiment, a reaction solution containing a plurality of test cells to which a calcium ion indicator is added is divided into three, a reaction solution containing a first test cell, and a reaction solution containing a second test cell. And the third test cell is obtained, the test substance is contacted with the first test cell, the test substance and the mitochondrial permeability enhancer are not contacted with the second test cell, The step of contacting the test cell with a mitochondrial membrane permeability enhancer. Further, a cytoprotective drug may be further contacted with each of the first to third test cells. The mitochondrial permeability enhancer is as described above.

  The third test cell can be used as a positive control because the amount of calcium ions in the reaction solution or in the cytoplasm is increased by the action of the mitochondrial membrane permeability enhancer. Therefore, in addition to comparing the calcium ion content measured for the first test cell and the second test cell, by comparing the calcium ion content measured for the first test cell and the third test cell. Therefore, more accurate evaluation can be performed.

  The screening method according to the present embodiment may perform evaluation based on the amount of active oxygen as well as evaluation based on the amount of calcium ions. That is, the screening method according to the present embodiment includes a step of adding an active oxygen indicator to a reaction solution containing the plurality of test cells in vitro, the first test cell, and the first test using the active oxygen indicator as an index. Measuring the amount of active oxygen for each of the two test cells and, if necessary, the third test cell, the first test cell, the second test cell, and, if necessary, the third test cell Comparing the amount of active oxygen measured for. Thereby, the inhibitory effect on the enhancement of mitochondrial membrane permeability related to cell death can be evaluated more accurately.

  Specific examples of the active oxygen indicator and the method of adding it are as described above. Moreover, the specific example of the measuring method of the amount of calcium ions and the amount of active oxygen is as above-mentioned.

  When the amount of active oxygen measured for the first test cell exceeds the amount of active oxygen measured for the second test cell, the test substance is evaluated as a substance that enhances mitochondrial membrane permeability. Here, from the viewpoint of further improving the accuracy of evaluation, the amount of calcium ions measured for the first test cells exceeded the amount of calcium ions measured for the second test cells, and was measured for the first test cells. When the amount of active oxygen exceeds the amount of active oxygen measured for the second test cell, it is preferable that the test substance is evaluated as a substance that enhances mitochondrial membrane permeability.

According to the screening method of the present embodiment, evaluation based on an initial signal of apoptosis induction due to enhanced mitochondrial membrane permeability (for example, an increase in the amount of Ca ²⁺ or O ₂ ⁻ in the cytoplasm) is possible. The screening method is also useful as a screening method for a substance that induces apoptosis.

  The test substance evaluated as a substance that enhances mitochondrial membrane permeability by the screening method of the present embodiment is suitably used as a mitochondrial membrane permeability enhancer. Since the mitochondrial membrane permeability enhancer can promote enhancement of mitochondrial membrane permeability, which is an initial signal for inducing apoptosis of cells, it can be suitably used, for example, as an apoptosis inducer. In addition, since cancer cell death can be induced through apoptosis-inducing action, it can also be suitably used as a therapeutic adjuvant in cancer cells that are resistant to anticancer agents or conventional anticancer agents.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to these examples.

[1. experimental method〕
(1) Cell culture Human neuroblastoma-derived SH-SY5Y cells were maintained in DMEM (Gibco) medium supplemented with 10% serum (FBS), 100 mg / mL penicillin and 100 U / mL streptomycin. Cells seeded in a flask (125 cm ² flask) were cultured in a 37 ° C., 5% CO ₂ incubator, and subculture was performed when the cells became 80-90% confluent (about 3 days). Cells transfected with SH-SY5Y cells and overexpressed Bcl-2 (Bcl-2 overexpressing cells) were similarly maintained.

(2) Addition of calcium ion indicator Cells maintained as described above were mechanically collected and centrifuged (1000 rpm, 3 minutes). The cells obtained as a centrifugal sediment were suspended in RH buffer (154 mM NaCl, 5.6 mM KCl, 10 mM Hepes, pH 7.4) and centrifuged again. Cells obtained as a centrifugal sediment were suspended in 5 mL of DMEM medium, and an intracellular fluorescent indicator Fluo-3 AM (1 mM in DMSO, DOJINDO) was added. The addition amount was 1 mM Fluo-3 AM 15 μl / 5 mL cell suspension. The cells were cultured for 30 minutes in a 37 ° C., 5% CO ₂ incubator to incorporate Fluo-3 AM into the cells.

(3) Preparation of cell suspension Cells treated with Fluo-3 AM were centrifuged, washed with RH buffer, and centrifuged again. Cells obtained as a centrifugal sediment were resuspended in 2 mL RH buffer. An equal amount of 0.4% Trypan-Blue solution was mixed with 20 μL of this cell suspension, and the number of cells was measured with a hemocytometer. Based on the measured number of cells, RH buffer was added to adjust the cell suspension concentration to 6 × 10 ⁵ cells / mL.

(4) Addition of active oxygen indicator and treatment with cytoprotective agent The cell suspension described above so that the total volume of the cell reaction solution when measuring the calcium ion amount and the active oxygen amount in (5) below is 1500 μL. And RH buffer were mixed. MCLA (25 μM, dissolved in RH buffer, Tokyo Kasei) 60 μL (final concentration is 1 μM) or CLA (25 μM, dissolved in RH buffer, Tokyo Kasei) 60 μL (final concentration is superoxide chemiluminescence indicator) 1 μM) was added. When a cytoprotective drug (100 μM cyclosporin A or 10 μM rasagiline) was added, 15 μL was added to the cell suspension together with MCLA. After the addition, the temperature was equilibrated at 37 ° C. for 7 minutes.

(5) Treatment with mitochondrial membrane permeability enhancer and measurement of calcium ion and active oxygen content The cell suspension equilibrated in temperature as described above was placed in a fluorescent cell (cuvette), and the fluorescence and chemistry shown in FIG. It set to the simultaneous measurement apparatus of light emission, and the measurement time was set (0.5 second / point x 1500 points). When a mitochondrial permeability enhancer (500-100 μMPK11195) was added, 15 μL was added at this stage. Table 1 shows an example of the composition of the reaction solution used for the measurement. The solvent is ethanol.

  The amount of calcium ions and the amount of active oxygen were measured by detecting fluorescence and chemiluminescence with the simultaneous measurement apparatus for fluorescence and chemiluminescence shown in FIG. In the measuring apparatus 100 shown in FIG. 5, the excitation light emitted from the LED light source 1 (500 nm) is irradiated to the cuvette 6 set in the block heater 5 through the bandpass filter 2, the opening 3 and the lens 4. . The reaction solution is contained in the cuvette 6. The fluorescence and chemiluminescence emitted from the reaction solution are guided to the detector 10 through the lens 7, the band blocking filter 8 and the shutter 9. The detector 10 quantifies the amount of fluorescence and chemiluminescence. The measuring apparatus 100 has the above-described configuration in the dark box 50.

[2. Increase of cytosolic Ca ²⁺ and O ₂ ⁻ content in the reaction solution by PK11195 (1)]
PK11195 was added as a mitochondrial membrane permeability enhancer to SH-SY5Y cells and Bcl-2 overexpressing cells, and the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution were measured according to the experimental method described above. The results are shown in FIG.

When PK11195 is added to SH-SY5Y cells (indicated as “II” in FIG. 1A, a curve indicated by PK11195 (CL) or PK11195 (FL)), the amount of Ca ²⁺ in the cytoplasm and in the reaction solution of O ₂ ^- increased amounts both were observed. On the other hand, there was no such increase in control SH-SY5Y cells to which PK11195 was not added (indicated as “I” in FIG. 1 (A). Curve indicated by Control (CL) or Control (FL)). . 1 to 4, “CL” means chemiluminescence, and “FL” means fluorescence.

On the other hand, Bcl-2 overexpressing cells do not add PK11195 (indicated as “II” in FIG. 1B, a curve represented by PK11195 (CL) or PK11195 (FL)). In the case (shown as “I” in FIG. 1B. Curve indicated by Control (CL) or Control (FL)), there is no increase in the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution. It was.

FIG. 1C is a graph showing an integrated value of Ca ²⁺ signals for 373 to 377.5 seconds in FIGS. 1A and 1B. FIG. 1 (D) is a graph showing the integrated value of the O ₂ ⁻ signal for 373 to 377.5 seconds in FIGS. 1 (A) and 1 (B). In SH-SY5Y cells (wild), the addition of PK11195 significantly increased both the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution. On the other hand, in Bcl-2 overexpressing cells (Bcl-2), there was no significant difference in the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution even when PK11195 was added.

Bcl-2 is known to be expressed in the outer mitochondrial membrane and prevent the increase in membrane permeability due to the opening of PTP. In addition, there is a report that overexpression of Bcl-2 in SH-SY5Y cells inhibits increase in membrane permeability and activation of apoptotic signals downstream thereof, thereby preventing cell death (J Neurochem, 2002). Year, 82 (4), pp. 913-923). Therefore, these results prove that the addition of PK11195 increases the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution through the opening of PTP.

[3. Increase of cytosolic Ca ²⁺ and O ₂ ⁻ amount in the reaction solution by PK11195 (2)]
In order to further prove that the increase in the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution due to the addition of PK11195 is through the opening of PTP, the effect of the addition of cyclosporin A (cytoprotective drug) was analyzed. . Cyclosporin A is known to inhibit the formation of PTP by inhibiting adenine nucleotide transferase (ANT) which is present in the inner mitochondrial membrane and constitutes PTP.

PK11195 as a mitochondrial membrane permeability enhancer and cyclosporin A as a cytoprotective agent were added to SH-SY5Y cells, and the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution were measured according to the experimental method described above. The results are shown in FIG.

When only PK11195 is added to SH-SY5Y cells (indicated as “II” in FIG. 2A, a curve indicated by PK11195 (CL) or PK11195 (FL)), the amount of Ca ²⁺ in the cytoplasm and the reaction solution An increase in the amount of O ₂ ⁻ was observed. There was no such increase in control SH-SY5Y cells to which neither PK11195 nor cyclosporin A was added (indicated as “IV” in FIG. 2 (A). Curve indicated by Control (CL) or Control (FL)). ). In addition, when both PK11195 and cyclosporin A are added to SH-SY5Y cells (indicated as “III” in FIG. 2A, a curve indicated by PK + CysA (CL) or PK + CysA (FL)), Ca in the cytoplasm. ^{Although both the 2+} amount and the O ₂ ⁻ amount in the reaction solution increased, the amount of Ca ²⁺ in the cytoplasm and the O ₂ ⁻ amount in the reaction solution were higher than when only PK11195 was added to SH-SY5Y cells. Diminished. In addition, when only cyclosporin A was added to SH-SY5Y cells, there was no significant increase in the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution (“I” in FIG. 2 (A)). (A curve indicated by CysA (CL) or CysA (FL).)

FIG. 2B is a graph showing the integrated value of the Ca ²⁺ signal for 373 to 377.5 seconds in FIG. FIG. 2C is a graph showing the integrated value of the O ₂ ⁻ signal for 373 to 377.5 seconds in FIG. When both PK11195 and cyclosporin A were added to SH-SY5Y cells, the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution were significantly reduced compared to the case where only PK11195 was added.

This result, together with the result of FIG. 1, shows that the increase in the amount of Ca ²⁺ in the cytoplasm observed this time is present in the mitochondrial Ca ²⁺ pool and is derived from Ca ²⁺ that has flowed into the cytoplasm at the opening of PTP. It was also clarified that the generation of reactive oxygen species (O ₂ ⁻ ) in the reaction solution was due to enhanced mitochondrial membrane permeability.

[4. Control of cytosolic Ca ²⁺ levels by rasagiline]
To the SH-SY5Y cells, PK11195 was added as a mitochondrial membrane permeability enhancer and rasagiline was added as a cytoprotective agent, and the amount of Ca ²⁺ in the cytoplasm was measured according to the above-described experimental method. The results are shown in FIG.

When only 500 μM, 250 μM, or 100 μM PK11195 was added to SH-SY5Y cells (indicated as “II”, “III”, and “IV” in FIG. 3A, respectively), there was an increase in the amount of Ca ²⁺ in the cytoplasm. It was seen. Control SH-SY5Y cells to which neither PK11195 nor rasagiline were added (indicated as “I” in FIG. 3A), and when 10 μM rasagiline alone was added to SH-SY5Y cells (in FIG. 3B, “ There was no such increase in "I". In addition, when both 500 μM PK11195 and 10 μM rasagiline were added to SH-SY5Y cells (indicated as “III” in FIG. 3B), and when both 250 μM PK11195 and 10 μM rasagiline were added (FIG. 3 (B ), Indicated as “V”), although an increase in the amount of Ca ²⁺ in the cytoplasm was observed, compared with the case where only PK11195 was added to SH-SY5Y cells (“II” in FIG. 3B). (Labeled 500 μM) or “IV” (250 μM)), the amount of Ca ²⁺ in the cytoplasm decreased. In FIG. 3B, the curve labeled “I” is for the case where only 10 μM rasagiline is added.

FIG. 3C is a graph showing the integrated value of the Ca ²⁺ signal for 373 to 377.5 seconds in FIGS. 3A and 3B. When both PK11195 and rasagiline were added to SH-SY5Y cells, the amount of Ca ²⁺ in the cytoplasm was significantly reduced compared to the case where only PK11195 was added. This decrease was also dependent on the concentrations of PK11195 and rasagiline.

  Rasagiline is a cell experiment using SH-SY5Y cells or other cultured cells, and also a substance that promotes PTP formation such as PK11195 in animal experiments, neurotoxin, oxidative stress, ischemia, neurotrophic factor by removing neurotrophic factors, etc. There are reports that nerve cells are protected from cell death (Non-patent Document 1, Curr Top Med Chem., 2012, 12 (20), pp. 2177-2188, Expert Rev Neurother., 13 (6), pp. 671-684).

[5. Screening for substances that suppress mitochondrial permeability
Using food ingredients sesamin (SM) and sesaminol (SML) as test substances, the mitochondrial membrane permeability-inhibiting activity was evaluated.

Pretreatment was performed in which SH-SY5Y cells were contacted with 100 μM sesamin (manufactured by Nagara Science Co., Ltd.) or sesaminol (manufactured by Nagara Science Co., Ltd.). Next, PK11195 was added as a mitochondrial membrane permeability enhancer, and the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution were measured according to the above-described experimental method. The results are shown in FIG.

When only PK11195 is added to SH-SY5Y cells (indicated as “I” in FIG. 4A, indicated as “I” in FIG. 4B), indicated by PK11195 (CL) or PK11195 (FL) Curve)), an increase in both the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution. When both PK11195 and sesaminol are added to SH-SY5Y cells (indicated as “II” in FIG. 4 (A), a curve indicated by PK11195 + SML (CL) or PK11195 + SML (FL)), and both PK11195 and sesamin (Indicated as “II” in FIG. 4B. Curve shown by PK + SM (CL) or PK + SM (FL).) In comparison with the case where only PK11195 is added to SH-SY5Y cells, The amount of Ca ^{2+ and the} amount of O ₂ ⁻ in the reaction solution decreased. In FIG. 4A, the curve indicated as SML (CL) or SML (FL) (indicated as “III”) is obtained when only sesaminol is added. Similarly, in FIG. 4B, the curve indicated as SM (CL) or SM (FL) (indicated as “III”) is obtained when only sesamin is added.

FIG. 4C is a graph showing the integrated value of the Ca ²⁺ signal for 373 to 377.5 seconds in FIGS. 4A and 4B. FIG. 4 (D) is a graph showing the integrated value of the O ₂ ⁻ signal for 373 to 377.5 seconds in FIGS. 4 (A) and 4 (B). The decrease in the amount of Ca ²⁺ in the cytoplasm and the amount of O ₂ ⁻ in the reaction solution by adding sesamin or sesaminol was statistically significant.

  This result indicates that sesamin and sesaminol have an action of suppressing the enhancement of mitochondrial membrane permeability.

  DESCRIPTION OF SYMBOLS 1 ... LED light source, 2 ... Band pass filter, 3 ... Opening part, 4 ... Lens, 5 ... Block heater, 6 ... Cuvette, 7 ... Lens, 8 ... Band block filter, 9 ... Shutter, 10 ... Detector, 50 ... Dark box, 100 ... measuring device.

Claims (18)

A method for evaluating mitochondrial membrane permeability,
Adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing test cells in vitro;
Using the calcium ion indicator as an index, measuring the amount of calcium ions permeating the mitochondrial membrane ;
Comparing the measured calcium ion amount with a first reference value,
The evaluation method, wherein when the measured calcium ion amount exceeds the first reference value, the mitochondrial membrane permeability of the test cell is evaluated to be enhanced. Adding an active oxygen indicator to the reaction solution in vitro;
Measuring the amount of active oxygen using the active oxygen indicator as an index;
Comparing the measured amount of active oxygen with a second reference value,
When the measured calcium ion amount exceeds the first reference value and the measured active oxygen amount exceeds the second reference value, it is evaluated that the mitochondrial membrane permeability of the test cell is enhanced. The evaluation method according to claim 1. The evaluation method according to claim 2, wherein the active oxygen is superoxide (O ₂ ⁻ ).   The evaluation method according to claim 2 or 3, wherein at least one of the calcium ion indicator and the active oxygen indicator is a fluorescent indicator.   The evaluation method according to claim 4, wherein the calcium ion indicator is a fluorescent indicator, and the active oxygen indicator is a chemiluminescent indicator.   The evaluation method according to any one of claims 1 to 5, wherein the enhancement of mitochondrial membrane permeability of the test cell indicates induction of apoptosis. A method for screening a substance that suppresses increased mitochondrial membrane permeability,
Adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing a plurality of test cells in vitro;
Dividing the reaction solution into at least two parts to obtain a reaction solution containing the first test cell and a reaction solution containing the second test cell, and increasing the permeability of the test substance and the mitochondrial membrane to the first test cell Contacting an agent and contacting the second test cell with a mitochondrial membrane permeability enhancer;
Measuring the amount of calcium ions permeating the mitochondrial membrane for each of the first test cell and the second test cell using the calcium ion indicator as an index;
Comparing the amount of calcium ions measured for the first test cell and the second test cell,
When the amount of calcium ions measured for the first test cell is less than the amount of calcium ions measured for the second test cell, the test substance is evaluated as a substance that suppresses enhancement of mitochondrial membrane permeability. Screening method. Adding an active oxygen indicator to a reaction solution containing the plurality of test cells in vitro;
Measuring the amount of active oxygen for each of the first test cell and the second test cell using the active oxygen indicator as an index;
Comparing the amount of active oxygen measured for the first test cell and the second test cell,
The amount of calcium ions measured for the first test cell is less than the amount of calcium ions measured for the second test cell, and the amount of active oxygen measured for the first test cell is the second test. The screening method according to claim 7, wherein the test substance is evaluated as a substance that suppresses enhancement of mitochondrial membrane permeability when the amount of active oxygen measured for cells is below. The screening method according to claim 8, wherein the active oxygen is superoxide (O ₂ ⁻ ).   The screening method according to claim 8 or 9, wherein at least one of the calcium ion indicator and the active oxygen indicator is a fluorescent indicator.   The screening method according to claim 10, wherein the calcium ion indicator is a fluorescent indicator, and the active oxygen indicator is a chemiluminescent indicator.   The screening method according to any one of claims 7 to 11, wherein the enhancement of mitochondrial membrane permeability of the test cell indicates induction of apoptosis. A screening method for substances that enhance mitochondrial membrane permeability,
Adding a calcium ion (Ca ²⁺ ) indicator to a reaction solution containing a plurality of test cells in vitro;
The reaction solution is divided into at least two to obtain a reaction solution containing the first test cell and a reaction solution containing the second test cell, contacting the test substance with the first test cell, Not contacting the test substance with the two test cells;
Measuring the amount of calcium ions permeating the mitochondrial membrane for each of the first test cell and the second test cell using the calcium ion indicator as an index;
Comparing the amount of calcium ions measured for the first test cell and the second test cell,
When the amount of calcium ions measured for the first test cell exceeds the amount of calcium ions measured for the second test cell, the test substance is evaluated as a substance that enhances mitochondrial membrane permeability. Screening method. Adding an active oxygen indicator to a reaction solution containing the plurality of test cells in vitro;
Measuring the amount of active oxygen for each of the first test cell and the second test cell using the active oxygen indicator as an index;
Comparing the amount of active oxygen measured for the first test cell and the second test cell,
The amount of calcium ions measured for the first test cell exceeds the amount of calcium ions measured for the second test cell, and the amount of active oxygen measured for the first test cell is the second test. The screening method according to claim 13 , wherein when the amount of active oxygen measured for a cell is exceeded, the test substance is evaluated as a substance that enhances mitochondrial membrane permeability. The screening method according to claim 14 , wherein the active oxygen is superoxide (O ₂ ⁻ ). The screening method according to claim 14 or 15 , wherein at least one of the calcium ion indicator and the active oxygen indicator is a fluorescent indicator. The screening method according to claim 16 , wherein the calcium ion indicator is a fluorescent indicator, and the active oxygen indicator is a chemiluminescent indicator. The screening method according to any one of claims 13 to 17 , wherein the enhancement of mitochondrial membrane permeability of the test cell indicates induction of apoptosis.