JP2011037722A - Prophylactic or inhibitive agent of neurocyte death caused by endoplasmic reticulum stress - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical that prevents or inhibits endoplasmic reticulum stress or neurocyte death caused by endoplasmic reticulum stress thereby to effectively prevent or treat diseases related to neurocyte death caused by endoplasmic reticulum stress while causes little side effects. <P>SOLUTION: The prophylactic or inhibitive agent for diseases related to neurocyte death caused by endoplasmic reticulum stress comprises yoku-kan san (yi-gan san, a Chinese herbal medicine) as an effective ingredient. The diseases related to neurocyte death caused by endoplasmic reticulum stress include Alzheimer disease, Parkinson disease, polyglutamine disease, mad cow disease, cerebral ischemia and amyotrophic lateral sclerosis. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a medicament for preventing or suppressing cell death caused by endoplasmic reticulum stress, particularly neuronal cell death. The present invention also relates to a medicament for preventing or treating neuronal cell death-related diseases caused by endoplasmic reticulum stress.

  The endoplasmic reticulum is a place where secretory proteins and membrane proteins are correctly folded and their three-dimensional structure is adjusted. In addition, the endoplasmic reticulum has a variety of physiological functions as a reservoir for intracellular calcium. However, physicochemical stresses such as ischemia, hypoxia, and gene mutations increase the protein that does not have a normal folding structure in the endoplasmic reticulum (unfolded protein) or cause calcium homeostasis (calcium homeostasis) in the endoplasmic reticulum. It is known that dysfunction of the endoplasmic reticulum is caused by being disturbed. Such a state in which the function of the endoplasmic reticulum is impaired and a state in which the function of the endoplasmic reticulum is impaired are referred to as endoplasmic reticulum stress.

  When endoplasmic reticulum stress is applied, cells immediately activate a defense system to avoid stress. On the endoplasmic reticulum membrane, there are membrane proteins PERK, IRE1α and ATF6 involved in stress signal transduction. These membrane proteins sense abnormal protein accumulation as an endoplasmic reticulum stress sensor and transmit signals into the cytoplasm or nucleus. For example, when PERK is activated, the α subunit (eIF2α) of translation initiation factor 2 is phosphorylated to suppress protein synthesis. In addition, activation of these stress sensors promotes transcription of molecular chaperones. This is a so-called endoplasmic reticulum stress response or unfolded protein response (UPR). However, when the state of strong endoplasmic reticulum stress continues, it has become clear that cells cannot resist stress and themselves select cell death (apoptosis).

  It has been pointed out that endoplasmic reticulum stress and cell death due to endoplasmic reticulum stress are involved in various diseases. For example, cell death due to endoplasmic reticulum stress is promoted in nerve cells in which a mutation in the causative gene (presenilin-1) of Alzheimer's disease is expressed. This is because the mutation of presenilin-1 suppresses the activation of the endoplasmic reticulum stress sensor, resulting in neuronal cell death. It has been reported that cell death under endoplasmic reticulum stress is also promoted by mutation of the causative gene presenilin-2 of Alzheimer's disease (Non-patent Document 1). Furthermore, refractory nerves such as Parkinson's disease, polyglutamine disease (Huntington's disease), mad cow disease (also called prion disease or bovine sea surface encephalopathy (BSE)), cerebral ischemia, amyotrophic lateral sclerosis (ALS), etc. It has been reported that neuronal cell death due to endoplasmic reticulum stress is also involved in the onset and progression of degenerative diseases. The number of patients with these intractable neurodegenerative diseases is increasing, and the development of effective prevention and treatment methods is desired.

  For example, as a therapeutic agent for Alzheimer's disease, a drug (acetylcholine degradation inhibitor) that prevents the degradation of acetylcholine and causes the activation of cerebral cortical neurons to delay the progression of dementia is used. However, such drugs cannot suppress neuronal cell death that is the cause of Alzheimer's disease. For this reason, although the symptoms of Alzheimer's disease improved, the cause could not be treated and could not be said to be a fundamental therapeutic agent. Further, based on the amyloid hypothesis, the development of suppression of amyloid β protein excision, immunotherapy for amyloid β protein, and the like has been underway. However, there is a problem of side effects, and further, desirable results are not obtained from the viewpoint of symptom improvement. Furthermore, there is currently no means for prevention or treatment of neuronal cell death after cerebral ischemia.

  Neurons cannot divide once they mature. Therefore, there is an urgent need to understand the onset mechanism of neuronal cell death due to endoplasmic reticulum stress and related diseases, and to develop therapeutic methods. However, no substance or component having an activity to effectively prevent or suppress endoplasmic reticulum stress or neuronal cell death due to endoplasmic reticulum stress has yet been reported.

Journal of Chemical Neuroanatomy 28 (2004) 67-78

  The present invention relates to a medicament capable of effectively preventing or treating neuronal cell death-related diseases caused by endoplasmic reticulum stress by preventing or suppressing endoplasmic reticulum stress or neuronal cell death caused by endoplasmic reticulum stress, and having few side effects. The purpose is to provide.

  The inventors of the present invention have made extensive studies to solve the above problems, and Yokukansan, which is a Chinese medicine (Chinese medicine), significantly prevents and suppresses neuronal cell death caused by sapigardine-induced endoplasmic reticulum stress. Based on this knowledge, we have conducted further research. As a result, the present inventors significantly prevent and suppress neuronal cell death under endoplasmic reticulum stress in the neuron expressing the mutation of the causative gene (presenilin 1) of Alzheimer's disease. I found. Yokukansan is a highly safe Kampo medicine that has been proven to have few side effects in conventional clinical use. In addition, there has been no effective therapeutic agent for preventing or suppressing neuronal cell death in cerebral ischemia. However, the present inventors have found that yokukansan is a small molecule induced by hypoxia (cerebral ischemia model). It was found that neuronal cell death due to endoplasmic reticulum stress is also significantly prevented and suppressed.

  Furthermore, among the constituent crude drugs of Yokukansan, the present inventors have also found that Kyukyu (Senkyu) is a neuronal cell death caused by endoplasmic reticulum stress in a neuronal cell in which the mutation of the causative gene for Alzheimer's disease (Presenilin 1) is expressed. Was found to be significantly prevented and suppressed. Kawakyu also found that neuronal cell death due to endoplasmic reticulum stress induced by hypoxia (cerebral ischemia model) was also significantly prevented and suppressed. In addition, it has been found that Shiba, one of the constituent herbal medicines of Yokukansan, tends to suppress neuronal cell death due to these endoplasmic reticulum stresses.

When a neuronal cell is induced to break calcium homeostasis or is exposed to a hypoxic state, expression of GRP78 / Bip or the like that protects the neuron cell is induced. On the other hand, induction of CHOP or the like that triggers cell death also occurs. When the balance between the two is lost and the expression of CHOP is dominant, the nerve cell is killed. For example, in a cell in which a mutation of a gene causing Alzheimer's disease (such as presenilin 1) is expressed, these balances are easily lost, and cell death is promoted. The present inventors have found that Yokukansan and its constituent crude drug Kawakyu have an action of increasing the expression of GRP78 / Bip, and this action suppresses cell death caused by endoplasmic reticulum stress. In addition, Yokukansan and its constituent crude drugs Kawakyu and Shibahu have the effect of suppressing the induction of CHOP, and this action has been found to suppress cell death due to endoplasmic reticulum stress.
From these findings, the present inventors, Yokukansan, and its constituent crude drugs Kawakyu and Shibahu are effective in preventing or suppressing neuronal cell death due to endoplasmic reticulum stress and endoplasmic reticulum stress in neurons, Furthermore, since there are few side effects, it came to be thought that it is useful for the prevention or treatment of the neuron death related disease by endoplasmic reticulum stress, such as Alzheimer's disease and cerebral ischemia.

Thus, a drug capable of preventing and suppressing neuronal cell death due to endoplasmic reticulum stress, for example, suppresses the developmental mechanism of Alzheimer's disease compared to an acetylcholine degradation inhibitor currently regarded as the only therapeutic agent for Alzheimer's disease. In that respect, it is a revolutionary medicine that is greatly different. In addition, Yokukansan, which is a crude drug, and Kawakyu and Saiko have few side effects, so they are safe even when administered prophylactically. For example, in familial Alzheimer's disease (family AD: FAD) families It can be suitably used for prevention of neuronal cell death-related diseases caused by endoplasmic reticulum stress, such as prevention of onset of undeveloped families.
The present inventors have further studied based on these findings, and have completed the present invention.
That is, the present invention includes the following inventions.

(1) A preventive or therapeutic agent for neuronal cell death-related diseases caused by endoplasmic reticulum stress, comprising yokukansan.
(2) The prevention according to (1), wherein the neuronal cell death-related disease caused by endoplasmic reticulum stress is Alzheimer's disease, Parkinson's disease, polyglutamine disease, mad cow disease, cerebral ischemia, or amyotrophic lateral sclerosis. Or a therapeutic agent.
(3) The preventive or therapeutic agent according to (1) or (2) above, wherein the neuronal cell death-related disease caused by endoplasmic reticulum stress is Alzheimer's disease or cerebral ischemia.

(4) A preventive or therapeutic agent for a neuronal cell death-related disease caused by endoplasmic reticulum stress, comprising as an active ingredient at least one herbal medicine selected from the group consisting of river cucumber and saiko.
(5) The preventive or therapeutic agent according to (4) above, wherein the crude drug is Kawakyu.
(6) The above (4) or (5), wherein the neuronal cell death-related disease caused by endoplasmic reticulum stress is Alzheimer's disease, Parkinson's disease, polyglutamine disease, mad cow disease, cerebral ischemia, or amyotrophic lateral sclerosis. The preventive or therapeutic agent according to 1.
(7) The preventive or therapeutic agent according to (5) or (6) above, wherein the neuronal cell death-related disease caused by endoplasmic reticulum stress is Alzheimer's disease or cerebral ischemia.

(8) An agent for preventing or suppressing neuronal cell death caused by endoplasmic reticulum stress, comprising yokukansan.
(9) A preventive or inhibitory agent for neuronal cell death caused by endoplasmic reticulum stress, comprising as an active ingredient at least one herbal medicine selected from the group consisting of river cucumber and saiko.
(10) Prevention or suppression of endoplasmic reticulum stress or cell death due to endoplasmic reticulum stress, comprising yokukansan or at least one herbal medicine selected from the group consisting of river kyu and shirahiko as an active ingredient Agent.

The present invention also provides
A method for preventing or treating neuronal cell death-related diseases caused by endoplasmic reticulum stress, wherein yokukansan is administered to a mammal;
A method for preventing or treating a neuronal cell death-related disease caused by endoplasmic reticulum stress, wherein at least one herbal medicine selected from the group consisting of river cucumber and saiko is administered to a mammal;
A method for preventing or suppressing neuronal cell death caused by endoplasmic reticulum stress, wherein yokukansan is administered to a mammal;
A method for preventing or suppressing neuronal cell death caused by endoplasmic reticulum stress, wherein at least one herbal medicine selected from the group consisting of river cucumber and shibahu is administered to a mammal;
Method of preventing or suppressing endoplasmic reticulum stress or cell death caused by endoplasmic reticulum stress by administering Yokukansan to mammal, and endoplasmic reticulum stress by administering at least one herbal medicine selected from the group consisting of Kawakyu and Shirahou to mammal Or a method for preventing or suppressing cell death due to endoplasmic reticulum stress,
About.

The present invention further provides the use of yokukansan for the manufacture of a preventive or therapeutic agent for neuronal cell death-related diseases caused by endoplasmic reticulum stress, and
The use of at least one herbal medicine selected from the group consisting of Kawasaki and Shiba for the manufacture of a preventive or therapeutic agent for neuronal cell death-related diseases caused by endoplasmic reticulum stress,
About.

  According to the present invention, endoplasmic reticulum stress and cell death of nerve cells and the like due to endoplasmic reticulum stress can be effectively prevented or suppressed because there are few side effects. Therefore, according to the present invention, neuronal cell death related to endoplasmic reticulum stress such as Alzheimer's disease, Parkinson's disease, polyglutamine disease, mad cow disease (BSE), cerebral ischemia and amyotrophic lateral sclerosis (ALS), etc. The disease can be prevented or treated effectively and safely. The prophylactic or therapeutic agent of the present invention is, for example, an epoch-making difference that is significantly different from the acetylcholine degradation inhibitor currently regarded as the only therapeutic agent for Alzheimer's disease in that it suppresses the developmental mechanism of Alzheimer's disease onset. Preventive or therapeutic agent.

FIG. 1 is a diagram showing the effect of yokukansan on cell death caused by sapsigargin (TG). FIG. 2 is a diagram showing the time-dependent effect of yokukansan on cell death caused by sapargardine (TG). FIG. 3 is a diagram showing the effect (concentration dependence) of yokukansan on cell death by sapargardine. 4A to 4D are fluorescence micrographs showing the effect (concentration dependence) of Yokukansan on cell death by sapargardine. FIG. 5 is a diagram showing the effect of yokukansan on cell death due to low oxygen load. FIGS. 6A to 6C are fluorescence micrographs showing the effect of yokukansan on cell death due to hypoxia. It is a figure which shows the result of the Western blot which investigated the effect | action of Yokukansan on the expression of GRP78 gene. It is a figure which shows a time-dependent change of the expression of GRP78 gene induced | guided | derived with tunicamycin (final concentration in a culture medium 5 microgram / mL). It is a figure which shows the time-dependent change of the expression of GRP78 gene induced | guided | derived with tunicamycin (final concentration in a culture medium 2 microgram / mL). It is a figure which shows the time-dependent change of the expression of the CHOP gene induced | guided | derived by tunicamycin (final concentration in a culture medium 5 microgram / mL). It is a figure which shows the time-dependent change of the expression of the CHOP gene induced | guided | derived by tunicamycin (final concentration in a culture medium 2 microgram / mL). It is a figure which shows the effect with respect to sapsigargin-induced cell death of each constituent crude drug of Yokukansan. It is a figure which shows the single toxicity of each constituent drug of Yokukansan. It is a figure which shows the effect of Yokukansan and each herbal medicine component with respect to the cell death by a sapigargin. It is a figure which shows the effect of Yokukansan and acupuncture with respect to the cell death by a sapigargin. It is a figure which shows the effect of yokukansan and shibahu with respect to the cell death by a sapigargin. It is a figure which shows the effect of Yokukansan and Kawakyu on the cell death by a sapisigargin. It is a figure which shows the effect of Yokukansan and each herbal medicine component with respect to the expression of GRP78 gene. It is a figure which shows the effect of Yokukansan and its constituent crude drug with respect to the expression of the GRP78 gene induced | guided | derived by sapigargin (TG). It is a figure which shows the effect of Yokukansan and its constituent crude drug with respect to the expression of the CHOP gene induced by sapisigargin (TG). It is a figure which shows the effect of Yokukansan and its constituent crude drug with respect to the expression of GRP78 gene induced by tunicamycin (Tm). It is a figure which shows the effect of Yokukansan and its constituent crude drug with respect to the expression of the CHOP gene induced by tunicamycin (Tm). It is a figure which shows the effect of Yokukansan with respect to the cell death by ER stress in Alzheimer's disease causative gene PS1 mutant cell ((DELTA) 9). It is a figure which shows the effect of the river stream with respect to the cell death by ER stress in the Alzheimer's disease causative gene PS1 mutant cell. It is a figure which shows the effect of Shiba on the cell death by ER stress in the Alzheimer's disease causative gene PS1 mutant cell. It is a figure which shows the effect of Yokukansan and Kawakyu on the expression of GRP78 gene. It is a figure which shows the effect of Yokukansan and Kawakyu on the expression of a CHOP gene.

The agent for preventing or treating neuronal cell death-related diseases caused by endoplasmic reticulum stress according to the present invention contains yokukansan.
Another aspect of the prophylactic or therapeutic agent of the present invention is the prevention of neuronal cell death-related diseases caused by endoplasmic reticulum stress, which contains as an active ingredient at least one herbal medicine selected from the group consisting of Kawakyu and Shiba. Or it is a therapeutic agent.

In the present invention, “prevention” refers to delaying or preventing the onset of a symptom or disease and its associated symptoms, preventing the subject from acquiring a symptom or disease, or the subject to a symptom or disease. Means reducing the risk of earning.
“Treatment” refers to the complete cure of a symptom or disease, the suppression of the progression and / or worsening of the symptom without complete cure, and the progression of the symptom or disease, or the It means to improve part or all and lead to the direction of healing.

  As Yokukansan, a commercially available Chinese medicine can be used. Commercially available Chinese herbal medicines include, for example, Tsumura Yokukansan Extract Granules (trade name) (manufactured by Tsumura), a Chinese herbal medicine, Osugi Yokukansan (trade name) (manufactured by Tokiwa Pharmaceutical Co., Ltd.); Preferred are pharmaceuticals such as Yuan Yokukansan (trade name, manufactured by Yuichi Pharmaceutical Co., Ltd.). Among them, TSUMURA Yokukansan Extract Granule (trade name) (manufactured by Tsumura Corporation), which is a medical herbal medicine, is preferable.

Cnidii Rhizoma, which is contained in the preventive or therapeutic agent of the present invention, is a herbal medicine used for Yokukansan, which is a Chinese medicine. Kawakyu is a rhizome of the plant nematode (scientific name: Cnidium officinale Makino) of the Umbelliferae family.
Saiko (Bupleuri Radix) is a herbal medicine used for yokukansan. Saiko is the root of the ciraceae Mishima Saiko (scientific name: Bupleurum scorzonerifolium, B. falcatum) or its variants.

  In the case where the preventive or therapeutic agent of the present invention contains at least one herbal medicine selected from the group consisting of river cucumber and saiko, it is preferable to contain at least river cucumber. By including the river cucumber, the effect is particularly high for prevention or treatment of neuron death related diseases such as Alzheimer's disease and cerebral ischemia.

  River cucumber and saiko used as active ingredients of the preventive or therapeutic agent of the present invention are both known ones, such as those marketed as herbal powders. In the present invention, commercially available crude drug powder and the like can be used as these crude drugs.

  Kawakyu and Shirahiko are usually included as an extract powder (dried extract) in traditional Chinese medicines (Chinese medicines) such as Yokukansan. As the preventive or therapeutic agent of the present invention, it is also preferable to use a Chinese herb medicine (Chinese medicine) provided by combining Kawayu and Saiko with a herbal medicine. As such a Chinese medicine, Yokukansan is preferable. The use of Yokukansan as a preventive or therapeutic agent of the present invention is one of the preferred embodiments of the present invention. An agent for preventing or treating neuronal cell death-related diseases caused by endoplasmic reticulum stress, including yokukansan, is one of the particularly preferred embodiments of the present invention. As the Yokukansan, those mentioned above are suitable.

  As described above, the preventive or therapeutic agent of the present invention contains yokukansan or contains at least one herbal medicine selected from the group consisting of Kawakyu and Shirahiko. The preventive or therapeutic agent of the present invention may or may not contain components other than yokukansan or at least one herbal medicine selected from the group consisting of Kawakyu and Shirahiko. As ingredients other than Yokukansan, Kawakyu and Shirahiko, pharmaceutically acceptable carriers and the like can be used.

  In the present invention, at least one kind of crude drug selected from the group consisting of Yokukansan or Kawakyu and Shirahiko can be used as it is or appropriately formulated as the preventive or therapeutic agent of the present invention. For example, the prophylactic or therapeutic agent of the present invention is known as Yokukansan, or at least one herbal medicine selected from the group consisting of Kawakyu and Shirahiko, and a pharmaceutically acceptable carrier optionally blended. It can be easily prepared by mixing, etc. by the above method.

  Examples of the dosage form of the preventive or therapeutic agent of the present invention include oral preparations such as solid preparations such as tablets, powders, granules and capsules; liquid preparations such as emulsions, syrups and suspensions. . Examples of preparations for parenteral administration include injections and drops. The dosage form of the preventive or therapeutic agent of the present invention is preferably an oral dosage form.

  As the pharmaceutically acceptable carrier in the present invention, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid formulations; solvents in liquid formulations, dissolution Adjuvants, suspending agents, isotonic agents, buffers, soothing agents and the like can be mentioned. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used.

Suitable examples of excipients include, for example, sugars or sugar alcohols such as lactose, corn starch, maltose, mannitol; starches or starch derivatives such as corn starch, dextrin, pregelatinized starch; celluloses such as crystalline cellulose and hydroxypropyl cellulose And cellulose derivatives; light anhydrous silicic acid and the like.
Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.

Preferable examples of the binder include crystalline cellulose, sucrose, mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like.
Preferable examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like.

Preferable examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.
Preferable examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

  Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; Examples thereof include hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol and the like.
Preferable examples of the buffer include buffer solutions such as phosphate, acetate, carbonate, citrate and the like.
Preferable examples of the soothing agent include benzyl alcohol.

Preferable examples of the preservative include paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Preferable examples of the antioxidant include sulfite and ascorbic acid.

  When the preventive or therapeutic agent of the present invention contains yokukansan, the content of yokukansan is, for example, about 1 to 99% by mass of the usual preparation as yokukansan extract powder if it is a solid preparation, Preferably it is about 5-90 mass%, More preferably, it is about 10-80 mass%.

  When the preventive or therapeutic agent of the present invention contains river cucumber, the content thereof is, for example, about 0.0001 to 99% by weight, preferably about 0.001 to 70% by weight. When the preventive or therapeutic agent of the present invention contains saiko, for example, if it is a solid preparation, the content thereof is about 0.0001 to 99% by weight of the usual preparation as the saiko extract powder, preferably about 0.001 to 50% by weight.

The administration method of the preventive or therapeutic agent of the present invention is preferably oral administration. The preventive or therapeutic agent of the present invention is more preferably administered orally before or between meals.
The dose and frequency of administration of the prophylactic or therapeutic agent of the present invention vary depending on age, body weight, dosage form, etc., but the dose for oral administration of a preparation containing yokukansan is usually yokukan The powdered powder is administered at a dose of about 10 μg to 500 mg, preferably about 100 μg to 300 mg per kg body weight. This amount is preferably administered orally in 1 to 3 divided doses per day. As a preparation containing Yokukansan, the above-mentioned TSUMURA Yokukansan extract granule (for medical use) (manufactured by Tsumura Corporation) is preferable.

  When an agent containing river cucumber is orally administered as a preventive or therapeutic agent of the present invention, it is usually about 1 μg to 50 mg, preferably about 10 μg to 30 mg per kg body weight, as an adult powder of river cucumber per day for adults. To be administered. In the case of orally administering an agent containing saiko, it is usually administered at an amount of about 1 μg to 50 mg, preferably about 10 μg to 30 mg per kg of body weight as an adult powder per day. This amount is preferably administered orally in 1 to 3 divided doses per day. Even when administering an agent containing river cucumber and salmon, it is preferable to administer the river cucumber and shihu.

  Since the preventive or therapeutic agent of the present invention has an action of effectively preventing or suppressing neuronal cell death caused by endoplasmic reticulum stress and endoplasmic reticulum stress in nerve cells, prevention or treatment of diseases related to neuronal cell death caused by endoplasmic reticulum stress. Can be suitably used. The neuronal cell death-related disease due to endoplasmic reticulum stress may be a disease in which neuronal cell death due to endoplasmic reticulum stress is involved in the onset or progression of the disease. The neuronal cell death-related diseases caused by endoplasmic reticulum stress in the present invention include Alzheimer's disease (familial Alzheimer's disease (FAD), Alzheimer-type senile dementia), Parkinson's disease, polyglutamine disease (Huntington's disease), and mad cow disease (bovine sponges). Encephalopathy (BSE)), cerebral ischemia, amyotrophic lateral sclerosis (ALS) and the like are preferable. Among these, the preventive or therapeutic agent of the present invention can be suitably used for the prevention or treatment of Alzheimer's disease or cerebral ischemia.

  As the administration target of the preventive or therapeutic agent of the present invention, mammals such as humans, mice, rats, rabbits, dogs, cats, cows, monkeys and pigs are preferable, and humans are more preferable. Moreover, as a subject of administration, a mammal that has developed or is likely to develop a neuronal cell death-related disease caused by endoplasmic reticulum stress is preferable. For example, by administering the preventive or therapeutic agent of the present invention from the initial stage when the above-mentioned neuronal cell death-related disease has developed, the symptom can be effectively improved or the progression of the symptom can be effectively prevented.

  Since the preventive or therapeutic agent of the present invention has few side effects, it can be suitably used for preventing the above-mentioned neuronal cell death-related diseases. For example, administration of the preventive or therapeutic agent of the present invention to a mammal or the like that may develop a neuronal cell death-related disease caused by endoplasmic reticulum stress before the onset of the disease delays or prevents the onset of the disease. be able to. Suitable mammals that may develop a neuronal cell death-related disease caused by endoplasmic reticulum stress include, for example, mammals (preferably humans) that may develop familial Alzheimer's disease (FAD).

  Since the preventive or therapeutic agent of the present invention contains at least one herbal medicine selected from the group consisting of Yokukansan or Kawakyu and Shibahu, it effectively prevents or suppresses endoplasmic reticulum stress in cells. It is something that can be done. Therefore, the preventive or therapeutic agent of the present invention can effectively prevent or suppress cell death due to endoplasmic reticulum stress.

An endoplasmic reticulum stress containing yokukansan or an agent for preventing or suppressing cell death due to endoplasmic reticulum stress is also one aspect of the present invention.
An endoplasmic reticulum stress or an agent that prevents or suppresses cell death due to endoplasmic reticulum stress, which contains at least one herbal medicine selected from the group consisting of river cucumber and saiko as an active ingredient is also one aspect of the present invention.
Since the preventive or suppressive agent of the present invention has the above-described action, it can be suitably used as a cell death protecting agent due to endoplasmic reticulum stress. The preventive or suppressive agent of the present invention and preferred embodiments thereof are the same as the preventive or therapeutic agent for neuronal cell death-related diseases caused by endoplasmic reticulum stress described above.

  “Inhibition of endoplasmic reticulum stress in (neural) cells” means to completely suppress endoplasmic reticulum stress in (neural) cells, or to suppress the progression of endoplasmic reticulum stress without complete inhibition. Means to stay. In addition, “suppression of (neuronal) cell death by endoplasmic reticulum stress” means that (neuronal) cell death due to endoplasmic reticulum stress is completely suppressed, or even if it is not completely suppressed (neural) It means to suppress the progress of cell death and to stop the progress.

  The preventive or suppressive agent of the present invention can be suitably used for, for example, prevention or suppression of endoplasmic reticulum stress in endocrine gland cells such as nerve cells, bone cells, pancreatic cells, or cell death due to endoplasmic reticulum stress. . Among them, it can be particularly preferably used for prevention or suppression of endoplasmic reticulum stress in neuronal cells or cell death of the cells due to endoplasmic reticulum stress.

An agent for preventing or suppressing neuronal cell death caused by endoplasmic reticulum stress containing yokukansan is also one aspect of the present invention.
An agent for preventing or suppressing neuronal cell death due to endoplasmic reticulum stress, which contains at least one herbal medicine selected from the group consisting of river cucumber and shiihu as an active ingredient, is also one aspect of the present invention.
The agent for preventing or inhibiting neuronal cell death according to the present invention and the preferred embodiment thereof are the same as the agent for preventing or treating neuronal cell death-related diseases caused by endoplasmic reticulum stress described above. The preventive or suppressive agent of the present invention can be suitably used as a protective agent for neuronal cell death caused by endoplasmic reticulum stress. The agent for preventing or suppressing neuronal cell death of the present invention is effective, for example, for preventing or suppressing neuronal cell death due to endoplasmic reticulum stress in patients with Alzheimer's disease, preventing or suppressing neuronal cell death due to endoplasmic reticulum stress due to cerebral ischemia, and the like. Is.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples at all.

The main reagents, cells and media used in this example are as follows.
As the Yokukansan, the Yokukansan extract preparation and trade name “Tsumura 54” manufactured by Tsumura Corporation were used. The Yokukansan extract preparation and trade name “Tsumura No. 54” manufactured by Tsumura Corporation used in the experiment are also simply referred to as “TJ-54”. Yokukansan's constituent herbal medicines (蒼朮 ジ ュ, 茯苓, 川, 当, 柴, licorice, 釣 鈎) A product made by Tsumura Corporation was used.
As thapsigargin, Sigma-Aldrich, and as tunicamycin, Sigma-Aldrich, were used. DMSO manufactured by Katayama Chemical Co., Ltd. was used.
The mouse neuroblastoma type Neuro2A cell (N2A) used was distributed from Osaka University Department of Psychiatry. When cultivating mouse neuroblastoma type Neuro2A cells, FCS (equivalent to fetal bovine serum and FBS) is added to Dulbecco's modified Eagle's medium (DMEM) so as to be 10% in the medium unless otherwise specified. Was performed at 37 ° C. using a medium (hereinafter referred to as 10% FCS-DMEM medium).
Human neuroblastoma SK-N-SH cells were obtained from RIKEN CellBank: RIKEN BioResource Center. Culture of human neuroblastoma SK-N-SH cells is FCS (equivalent to fetal bovine serum, FBS) so as to be 10% in the medium in α-MEM medium unless otherwise specified. Was carried out at 37 ° C. using a medium supplemented with (hereinafter referred to as FCS-added MEM medium).

<Example 1>
In general, endoplasmic reticulum stress is induced by stress such as nutrient starvation, ischemia, hypoxia and gene mutation, and this endoplasmic reticulum stress is tunicamycin (hereinafter also simply referred to as “Tm”), sapisigargin (thapsigargin). Hereinafter, it can also be artificially induced into cultured cells by administration of “TG”).
In Example 1, the influence of Yokukansan on cell death caused by sapigargin was examined. That is, endoplasmic reticulum stress (ER stress) was induced in cells by administration of sapigargin, and the influence of yokukansan on cell death due to the endoplasmic reticulum stress was examined.
TJ-54 (Yokukansan extract preparation) was added to the mouse neuroblastoma type Neuro2A cells (N2A) in 10% FCS-DMEM medium to a final concentration of 400 μg / mL. 1.5 hours after the addition of TJ-54, sapsigargin (500 μM in DMSO) dissolved in DMSO was added to the medium to a final concentration of 1 μM. To the culture medium of the control (control) group of mouse neuroblastic cell types Neuro2A cells, TJ-54 and sapigargin were not added, but DMSO used as a solvent was added in an amount of 1/500 of the medium volume (volume). Cell death was evaluated by the method described below.

(Evaluation of cell death)
For the evaluation of cell death, at least 300 cells were counted from four independent visual fields, and among them, Hoechst33342 (Hoechst33342 bis-benzamide (trade name, Sigma-Aldrich) 10 μM and propidium iodide (PI) (Sigma-Aldrich) added at 10 μM, Hoechst positive (blue fluorescence is emitted), PI positive (red fluorescence is emitted), and nuclear fragmentation Determine the number of cells that have been killed, and the percentage of cell death in the measured cells (the percentage of cells that have undergone cell death relative to the whole measured cell, hereinafter also referred to as “the percentage of cell death”) Based on the cell death rate in the control group (DMSO Y = 0), the rate of cell death (cell death rate) in each group (cell death rate) was calculated. Experiment Was performed three times, and a significant difference with respect to the control group was obtained by statistical processing by student's-t test (* P <0.05).

As a result, TJ-54 suppressed cell death after 6.5 hours of TG stimulation by nearly 50% as compared with the control group by pretreatment with 400 μg / mL (FIGS. 1 and 2).
In FIG. 1, DMSO is a DMSO addition group, Y-0 is no Yokukansan (TJ-54) addition, Y-400 is a Yokukansan (400 μg / mL) addition group, and TG is a sapishigargin addition group. is there. That is, (A) DMSO Y-0 in FIG. 1 is a control group. (B) DMSO Y-400 is a group in which DMSO is added after adding 400 μg / mL of Yokukansan to the medium. (C) TG Y-0 is a group to which TG was added without adding Yokukansan. (D) TG Y-400 is a group in which TG was added after adding 400 μg / mL of Yokukansan to the medium. The result shown in FIG. 1 is the ratio of cell death in each group with respect to the control group 6.5 hours after TG stimulation (addition) (when the control group is 1).
Furthermore, the effect of yokukansan after the above TG stimulation (addition) was examined in a time-dependent manner. The result is shown in FIG. In FIG. 2, rhombus (◇) is a group to which TG was added without adding Yokukansan ((C) in FIG. 1). A square (□) is a group (Y in FIG. 1) to which TG was added after adding 400 μg / mL of Yokukansan to the medium.

<Example 2>
In the same manner as in Example 1, endoplasmic reticulum stress (ER stress) was induced in cells by administration of sapsigargin, and the influence of yokukansan on cell death due to the endoplasmic reticulum stress was examined.
Mouse neuroblastoma Neuro2A cells (N2A) in 10% FCS-DMEM medium, TJ-54 (Yokukansan extract preparation) at a final concentration of 50 μg / mL, 200 μg / mL, 400 μg / mL, or 800 μg / mL It added to the culture medium so that it might become. 1.5 hours after the addition of TJ-54, sapsigargin (500 μM in DMSO) dissolved in DMSO was added to the medium to a final concentration of 1 μM. To a control (control) group of neuroblastoma Neuro2A cells, TJ-54 and sapishargine were not added, but DMSO used as a solvent was added in an amount of 1/500 of the medium amount (volume). Cell death was evaluated in the same manner as in Example 1. The results are shown in FIGS. FIG. 3 shows the ratio of cell death to the concentration of Yokukansan (TJ-54) in the medium. Y-0 to Y-800 means the case where the TJ-54 concentration in the medium is 0 to 800 μg / mL, respectively. FIG. 4 is a fluorescence micrograph of N2A cells 6.5 hours after TG addition. 4A shows the case where TJ-54 was not added to the medium (Yokukansan in the medium 0 μg / mL), and FIG. 4B shows the case where TJ-54 was added to the medium at a final concentration of 50 μg / mL. (C) shows the case where TJ-54 is added to the medium at a final concentration of 200 μg / mL, and (D) shows the case where TJ-54 is added to the medium at a final concentration of 800 μg / mL. In the fluorescence micrograph, the portion that appears blue or red (white or gray in the gray scale photo) is a cell in which cell death has occurred.
3 and 4, TJ-54 suppressed cell death after 6.5 hours of TG stimulation (addition) in a dose-dependent manner up to a concentration of 400 μg / mL in the medium, compared to the control group, but it was excessive at 800 μg / mL. On the contrary, Yokukansan itself showed a cell death promoting effect.

<Example 3>
ER stress is also induced by a low oxygen load. In Example 3, the effect of Yokukansan on cell death due to hypoxia was examined.
TJ-54 (Yokukansan extract preparation) was added to the mouse neuroblastoma type Neuro2A cells (N2A) in 10% FCS-DMEM medium to a final concentration of 400 μg / mL. 1.5 hours after the addition of TJ-54, the mouse neuroblastoma type Neuro2A cells were placed in a hypoxic chamber and subjected to hypoxic stimulation for 6 hours (hypoxia (6 hours) + yokukansan (400 μg / mL)). . As a comparison, mouse neuroblast cell type Neuro2A cells were not added with Yokukansan, but placed in a hypoxic chamber and subjected to hypoxic stimulation for 6 hours (hypoxia (6 hours)). In the same manner as in Example 1, in each group based on the cell death rate in cells (control: (A) normoxic pressure) cultured with normoxia (normal oxygen concentration) without adding TJ-54 Cell death rate was calculated. The same experiment was performed three times, and a significant difference with respect to the control group was obtained by performing statistical processing by student's-t test (* P <0.05). The results are shown in FIGS.

FIG. 5 is a diagram showing the effect of yokukansan on cell death due to low oxygen load. FIG. 6 (A) shows mouse neuroblastoma type Neuro2A cells (control) in which TJ-54 was not added and placed under normoxia; (B) shows mouse neuroblastoma species Neuro2A to which hypoxic stimulation was applied for 6 hours. Cells (without TJ-54 added); (C) shows a fluorescence microscope of mouse neuroblastoma type Neuro2A cells to which TJ-54 was added to a final concentration of 400 μg / mL and then subjected to hypoxic stimulation for 6 hours. It is a photograph. In the fluorescence micrograph of FIG. 6, a portion that appears blue or red (white or gray in a grayscale photo) is a cell in which cell death has occurred. 6A to 6C, it can be seen that in (C), fewer cells have undergone cell death than in (B).
As can be seen from FIGS. 5 and 6, TJ-54 suppressed cell death 6 hours after hypoxic stimulation compared with the hypoxic group by pretreatment with 400 μg / mL.

<Example 4>
In order to examine the reason why Yokukansan showed a protective effect against ER stress by sapisigargin, the expression level of GRP78, a chaperone, was examined in N2A cells (mouse neuroblastoma cell type Neuro2A cells).
The N2A cell culture solution was divided into the Yokukansan addition group and the non-addition group. Yokukansan was added to the Yokukansan group so that the culture medium concentration was 400 μg / mL. Each cell was collected 6 hours after the addition of Yokukansan, and Western blotting was performed according to a conventional method. The GRP78 antibody used was anti-Bip mAb (Cell Signaling Tchnology, Beverly, MA), and HRP-conjugated anti-mouse IgG Ab (Cell Signaling Tchnology, Beverly, MA) was used as the secondary antibody.
The result of the Western blot is shown in FIG. In FIG. 7, Y- is the case of 10% FCS-DMEM medium without yokukansan. Y + is a case where 400 μg / mL of Yokukansan is added to 10% FCS-DMEM medium. As is clear from FIG. 7, in the cells administered with Yokukansan (TJ-54), increased expression of GRP78 was observed. This result suggested the effect of increasing GRP78 expression by yokukansan.

<Example 5>
Using tunicamycin (Tm) as an ER stress inducer, the effect of TJ-54 on the expression of GRP78 (BiP), one of ER stress sensors, was examined.
TJ-54 (Yokukansan extract preparation) was added to the human neuroblastoma SK-N-SH cells in the FCS-added MEM medium so that the final concentration was 700 μg / mL. 24 hours after the addition of TJ-54, tunicamycin (Tm) dissolved in distilled water (2 mg or 5 mg / mL in distilled water) was added to the medium so that the final concentration was 2 μg / mL or 5 μg / mL. TJ-54 and Tm were not added to the cells in the control group, but distilled water used as a solvent was added in an amount of 1/1000 of the medium volume (volume). Cells were recovered at 0, 6 and 24 hours after addition of Tm, and total RNA was recovered with an RNAeasy kit (Qiagen). Using this RNA as a template, cDNA was prepared by SuperScript (registered trademark) II Reverse Transcriptase (trade name, manufactured by Invitrogen). Next, PCR (95 ° C., 0.5 min) using this cDNA as a template and GRP78 amplification primer 5′-ga aaggatggtt aatgatgctg ag-3 ′ (SEQ ID NO: 1) and 5′-gtcttcaatgtcagcatct-3 ′ (SEQ ID NO: 2) , 55 ° C for 1 minute, 72 ° C for 1 minute for 30 cycles). The obtained PCR product was used for agarose electrophoresis to evaluate the expression of GRP78 mRNA. GAPDH expression was used as a control as an internal standard. The results of agarose electrophoresis are shown in FIGS. 8 and 9 show the results of examining the expression of the GRP78 gene at each elapsed time after adding tunicamycin (Tm) to the medium at a final concentration of 5 μg / mL (FIG. 8) or 2 μg / mL (FIG. 9). 8 and 9, C is a control (not treated with Tm), and TJ is TJ-54 added to the medium at 700 μg / mL. The time in FIGS. 8 and 9 is the elapsed time (0, 6 and 24 hours) since Tm was added to the medium. In addition, 24 hours before Tm addition, TJ-54 of the said density | concentration is added to the culture medium.

  8 and 9, the expression of GRP78 was increased compared to the TJ-54 non-added group even in the absence of Tm stimulation (addition) at 24 hours after the addition of TJ-54. On the other hand, in the TJ-54 non-added group (control cells: C), GRP78 expression was remarkably increased 6 hours and 24 hours after stimulation (addition) with Tm of 5 μg / mL. In the TJ-54 added group, the GRP78 expression level increased about twice as much in 6 hours as compared to the GRP78 expression level in 0 hours, but after 24 hours, the GRP78 expression before Tm stimulation in the TJ-54 non-added group was observed. It recovered to the level (Fig. 8). The same tendency was also observed in the Tm 2 μg / mL stimulation (addition) group (FIG. 9). From these results, it was found that the addition of yokukansan increased the expression of GRP78 at the transcription level even in the absence of ER stress.

<Example 6>
Using tunicamycin (Tm) as an ER stress inducer, the effect of TJ-54 on the expression of CHOP (GADD153), one of ER stress sensors, was examined.
TJ-54 (Yokukansan extract preparation) was added to the human neuroblastoma SK-N-SH cells in the FCS-added MEM medium so that the final concentration was 700 μg / mL. 24 hours after the addition of TJ-54, tunicamycin (Tm) dissolved in distilled water (2 mg / mL or 5 mg / mL in distilled water) was added to the medium so that the final concentration was 2 μg / mL or 5 μg / mL. . Yokukansan and Tm were not added to the cells in the control group, but distilled water used as a solvent was added in an amount of 1/1000 of the medium amount (volume). Cells were recovered at 0, 6 and 24 hours after addition of Tm, and total RNA was recovered with an RNAeasy kit (Qiagen). Using this RNA as a template, cDNA was prepared by SuperScript (registered trademark) II Reverse Transcriptase (trade name, manufactured by Invitrogen). Next, PCR was performed under the same conditions as in Example 5 using this cDNA as a template and CHOP amplification primers 5'-catacaccaccacacctgaaag-3 '(SEQ ID NO: 3) and 5'-ccgtttcctagttcttccttgc-3' (SEQ ID NO: 4). went. The obtained PCR product was used for agarose electrophoresis to evaluate the expression of CHOP mRNA. GAPDH expression was used as a control as an internal standard. The results are shown in FIGS.

  10 and 11 show the results of examining the expression of the CHOP gene at each elapsed time after adding tunicamycin (Tm) to the medium at a final concentration of 5 μg / mL (FIG. 10) or 2 μg / mL (FIG. 11). 10 and 11, C is a control (not treated with Tm), and TJ is obtained by adding TJ-54 to a medium at 700 μg / mL. The time in FIGS. 10 and 11 is the elapsed time (0, 6 and 24 hours) after Tm was added to the medium. In addition, 24 hours before Tm addition, TJ-54 of the said density | concentration is added to the culture medium.

  10 and 11, when TJ-54 was added, the expression of CHOP slightly increased compared to the TJ-54 non-added group in the absence of Tm stimulation (addition) at 24 hours after the addition of TJ-54. Was. On the other hand, in the TJ-54 non-added group (control cells: C), CHOP expression was remarkably increased 6 hours and 24 hours after stimulation with Tm of 5 μg / mL. In the TJ-54 addition group, the CHOP expression level increased by about 2 times in 6 hours compared with the CHOP expression level in 0 hours, but after 24 hours, the CHOP expression level had recovered to the level before the Tm stimulation. (FIG. 10). In the Tm 2 μg / mL stimulation (addition) group, the expression of CHOP 6 hours after stimulation was similar to that before stimulation (FIG. 11).

From the results of Examples 1 to 6, Yokukansan (TJ-54) has a cell death suppression (cell protection) effect on ER stress stimulation such as TG, Tm, and hypoxia stimulation, and this cell death suppression. The effect was found to be due to the action of yokukansan to increase GRP78 expression (FIGS. 8 and 9). In addition, when TJ-54 was added, the expression of CHOP having a cell death inducing action was weaker than that of GRP78, and the increase of CHOP after ER stress stimulation was higher than that of the group without TJ-54 addition. The TJ-54 addition group was lower (FIGS. 10 and 11). For this reason, normally, in cells under ER stress, the expression of CHOP is increased similarly to GRP78, and as a result, cell death is induced. However, in cells to which Yokukansan has been added in advance, the cells are not under ER stress. Even in this state, GRP78 expression increased at the transcriptional level, and CHOP expression was lower than that of GRP78, suggesting that cell death was suppressed.
Next, it was investigated which constituent crude drug of Yokukansan (TJ-54) contributed to these cell death inhibitory actions.

<Example 7>
The effect of each constituent crude drug of TJ-54 on TG-induced cell death was examined.
Mouse neuroblastoma Neuro2A cells (N2A) in 10% FCS-DMEM medium were treated with TJ-54 (Yokukansan extract preparation: G8 in FIG. 12) or 7 types of Yokukansan constituent herbal medicines (Bukuryu), Kawakyu (Senkyu), Toki (Saiki), Shiba (Psycho), Licorice (Ganzo), and Chotou (Choutokou)) so that the final concentration is 200 μg / mL. Added to. 1.5 hours after the addition of TJ-54 or herbal medicine, sapigargin (TG) dissolved in DMSO (500 μM in DMSO) was added to the medium to a final concentration of 1 μM. To the cells of the control group (control), TJ-54, constituent crude drugs and TG were not added, but DMSO used as a solvent was added in an amount of 1/500 of the medium amount (volume).

(Evaluation of cell death)
The evaluation of cell death was performed by measuring at least 300 cells from 4 independent visual fields, and in that, after 20 hours of TG loading (addition), Hoechst33342 (trade name, Hoechst33342 bis-benzamide, Sigma-Aldorich) 10 μM and propidium iodide (PI) (Sigma-Aldorich) 10 μM were added, and Hoechst-positive, PI-positive, and nuclear fragmented cells were determined as cells undergoing cell death, Calculate the percentage of cell death (measured percentage of cell death) in the measured cells, and use the percentage of cell death in G0 (control: no TG added, neither yokukansan nor constituent crude drugs) As a result, the ratio of the number of cells in which cell death occurred in each group (cell death number) (cell death ratio) was calculated. The same experiment was performed three times, and a significant difference with respect to the G0 group was obtained by performing statistical processing by student's t test (* P <0.05).

The results are shown in FIG. The description in FIG. 12 is as follows.
G0: Control (no TG added, neither yokukansan nor constituent herbal medicines), G1: ジ ュ (Sojitsu), G2: 茯苓 (Bukuryu), G3: Kawakyu (Senkyu), G4: Toki, G5: Saiko, G6: Licorice, G7: Chotou, G8: Yokukansan (TJ-54).
Each crude drug was added to the medium at 200 μg / mL. The loading time of TG is 20 hours.
From FIG. 12, the percentage of cell death 20 hours after addition of TG was significantly lower than that of G0 (control) for moth (G2), Kawakyu (G3), and Toki (G4). A cell death promoting action was observed for Chotoko (G7).

<Example 8>
The single toxicity of each herbal medicine included in Yokukansan was examined.
Mouse neuroblastoma Neuro2A cells (N2A) in 10% FCS-DMEM medium were treated with TJ-54 (Yokukansan extract preparation: labeled as Yokukansan in FIG. 13) or 7 types of Yokukansan constituent crude drugs (蒼朮 ( Sojutsu), 茯苓 (Bakuryo), Kawakyu (Senkyu), Toki (Saiki), Shiba (Psycho), Licorice (Ganzo), and Chotou (Choutokou)) at a final concentration of 200 μg / mL It added to the culture medium so that it might become. The extent of cell death 24 hours after the addition of TJ-54 or herbal medicine was evaluated. Cell death was evaluated in the same manner as in Example 7. The percentage of cell death in each group was calculated based on the percentage of cell death (ratio of cells that caused cell death in all cells) of the control (no yokukansan or component crude drug added). . The same experiment was performed three times, and a significant difference with respect to the control group was obtained by performing statistical processing by student's-t test (* P <0.05).
As a result, the rate of cell death 24 hours after addition of licorice or Chotou was significantly higher than that of the control, and a cell death promoting effect was observed for these herbal medicines. In other words, licorice and Chotomochi were each significantly shown to be toxic to neurons.
The results are shown in FIG. In FIG. 3 and Table 1, “no crude drug” is the control.

<Example 9>
By WTS-1 assay, the effect of each crude drug component on cell death due to TG loading in human neuroblastoma SK-N-SH cells was examined.
Human neuroblastoma SK-N-SH cells in MEM medium with FCS added to TJ-54 (Yokukansan extract preparation), or 7 types of Yokukansan herbal medicines (Yujutsu, Bukuryu), Kawakyu (Senkyu), Toki (Toki), Shiba (Psycho), Licorice (Ganzo), and Chotou (Chrysanthemum)) were added at a final concentration (0 to 200 μg / mL) shown in FIG. . 1.5 hours after the addition of TJ-54 or herbal medicine, sepsigardine (500 μM in DMSO) dissolved in DMSO was added to the medium to a final concentration of 3 μM. The control (control) group was not added with herbal medicines or sapigargin, but DMSO used as a solvent was added in an amount of 1/500 of the medium amount (volume). The cell death was evaluated by expressing the culture supernatant 3 hours after TG stimulation and using the WST-1 assay kit (manufactured by Dojindo) as the inhibition rate against cell death in the control group to which only the solvent was added. The same experiment was performed three times, and a significant difference with respect to the control group was obtained by performing statistical processing by student's-t test (* P <0.05). As a result, Yokukansan (Yokukansan), Senkyu (Ryukyu) and Psycho (Shibahu) significantly suppressed cell death 3 hours after TG stimulation (addition). The results are shown in FIG. Moreover, the extract from FIG. 14 is shown to FIGS. 15-17 about the constituent crude drug by which the especially favorable cell death suppression effect was recognized. 15 to 17, the two components of Shibahu and strawberry showed a tendency similar to Yokukansan at a medium concentration of 100 μg / mL. Furthermore, when the medium concentration was 200 μg / mL, Saihu and Kawakyu showed remarkable effects.

<Example 10>
It was investigated whether the constituent crude drugs included in Yokukansan affect the expression of GRP78.
In the culture medium of human neuroblastoma SK-N-SH cells, TJ-54 (Yokukansan extract preparation) is at a final concentration of 200 μg / mL, and each constituent crude drug is at a final concentration of 200 μg / mL. 54 or herbal medicine was added to the medium. Each cell was recovered 6.5 hours after the addition, and Western blotting was performed according to a conventional method. The GRP78 antibody used was anti-Bip mAb (Cell Signaling Tchnology, Beverly, MA), and HRP-conjugated anti-mouse IgG Ab (Cell Signaling Tchnology, Beverly, MA) was used as the secondary antibody.
As a result, Akira Choto and Yokukansan markedly increased GRP78 expression. Other components also tended to increase the expression of GRP78 except for licorice. The results are shown in FIG.

<Example 11>
The effects of Yokukansan and each constituent crude drug on TG stimulation were examined.
The human neuroblastoma SK-N-SH cells in MEM medium supplemented with FCS have a final concentration of 800 μg / mL for TJ-54 (yokukansan extract preparation) and a final concentration of 200 μg / mL for each constituent crude drug. TJ-54 or herbal medicine was added to the medium. 1.5 hours after the addition of TJ-54 or herbal medicine, sapigargin (TG) (1 mM in DMSO) dissolved in DMSO was added to the medium to a final concentration of 2 μM. To the control group, none of TJ-54, herbal medicine, and sapigargin was added, and DMSO used as a solvent was added in an amount of 1/500 of the medium amount (volume). Cells were collected 6 hours after TG addition, and total RNA was collected using RNAeasy kit (Qiagen). Using this RNA as a template, cDNA was prepared by SuperScript (registered trademark) II Reverse Transcriptase (trade name, manufactured by Invitrogen). Next, GRP78 amplification primers 5′-ga aaggatggtt aatgatgctg ag-3 ′ (SEQ ID NO: 1) and 5′-gtcttcaatgtcagcatct-3 ′ (SEQ ID NO: 2) were used under the same conditions as in Example 5 using this cDNA as a template. PCR was performed. The obtained PCR product was used for agarose electrophoresis to evaluate the expression of GRP78 mRNA. GAPDH expression was used as a control as an internal standard. As a result, significant suppression of GRP78 expression was observed in the TJ-54 constituent crude drug group pretreated with Saiko or Senkyu. The results are shown in FIG. In FIG. 19 and FIGS. 20 to 22 described below, (−) indicates that TG (or Tm) stimulation (addition) was not performed. (+) Indicates that TG (or Tm) stimulation (addition) was performed.

<Example 12>
The human neuroblastoma SK-N-SH cells in MEM medium supplemented with FCS have a final concentration of 800 μg / mL for TJ-54 (yokukansan extract preparation) and a final concentration of 200 μg / mL for each constituent crude drug. Then, 1.5 hours after adding TJ-54 or herbal medicine to the medium, sapigargin (TG) dissolved in DMSO (500 μM in DMSO) was added to the medium to a final concentration of 2 μM. To the control group, none of TJ-54, herbal medicine, and sapigargin was added, and DMSO used as a solvent was added in an amount of 1/500 of the medium amount (volume). Cells were collected 6 hours after TG addition, and total RNA was collected using RNAeasy kit (Qiagen). Using this RNA as a template, cDNA was prepared by SuperScript (registered trademark) II Reverse Transcriptase (trade name, manufactured by Invitrogen). Subsequently, PCR was carried out under the same conditions as in Example 5 using CHOP amplification primers 5'-catacaccaccacacctgaaag-3 '(SEQ ID NO: 3) and 5'-ccgtttcctagttcttccttgc-3' (SEQ ID NO: 4) using this cDNA as a template. went. The obtained PCR product was used for agarose electrophoresis to evaluate the expression of CHOP mRNA. GAPDH expression was used as a control as an internal standard. As a result, suppression of induction of CHOP expression was observed in the TJ-54 constituent crude drugs in the group pretreated with Saiko, Senkyu, or Toki. The results are shown in FIG.

<Example 13>
The human neuroblastoma SK-N-SH cells in MEM medium supplemented with FCS have a final concentration of 800 μg / mL for TJ-54 (yokukansan extract preparation) and a final concentration of 200 μg / mL for each constituent crude drug. TJ-54 or herbal medicine was added to the medium. After 1.5 hours from the addition of TJ-54 or crude drug, tunicamycin (Tm) dissolved in distilled water (2 mg / mL in distilled water) was added to the medium to a final concentration of 2 μg / mL. To the control group, none of TJ-54, herbal medicine and tunicamycin was added, and distilled water used as a solvent was added in an amount of 1/1000 of the medium volume (volume). Cells were collected 6 hours after the addition of Tm, and total RNA was collected using an RNAeasy kit (Qiagen). Using this RNA as a template, cDNA was prepared by SuperScript (registered trademark) II Reverse Transcriptase (trade name, manufactured by Invitrogen). Next, using this cDNA as a template, the same conditions as in Example 5 were used, using GRP78 amplification primers 5'-ga aaggatggtt aatgatgctg ag-3 '(SEQ ID NO: 1) and 5'-gtcttcaatgtcagcatct-3' (SEQ ID NO: 2). PCR was performed. The obtained PCR product was used for agarose electrophoresis to evaluate the expression of GRP78 mRNA. GAPDH expression was used as a control as an internal standard. As a result, weak suppression of GRP78 expression was observed in the TJ-54 constituent crude drugs pretreated with Saiko (Saiko) or Chotou (Choutokou), but compared to the response observed after TG stimulation. The effect was weak. The results are shown in FIG.

<Example 14>
The human neuroblastoma SK-N-SH cells in the FCS-added MEM medium have a final concentration of 800 μg / mL for TJ-54 (Yokukansan extract preparation) and a final concentration of 200 μg / mL for each constituent crude drug. TJ-54 or herbal medicine was added to the medium. 1.5 hours after addition of TJ-54 or crude drug, tunicamycin (Tm) dissolved in distilled water (2 mg / mL in distilled water) was added to the medium so that the final concentration was 2 μg / mL. To the control group, none of TJ-54, herbal medicine and tunicamycin was added, and distilled water used as a solvent was added in an amount of 1/1000 of the medium volume (volume). Cells were collected 6 hours after the addition of Tm, and total RNA was collected using an RNAeasy kit (Qiagen). Using this RNA as a template, cDNA was prepared by SuperScript (registered trademark) II Reverse Transcriptase (trade name, manufactured by Invitrogen). Subsequently, PCR was carried out under the same conditions as in Example 5 using CHOP amplification primers 5'-catacaccaccacacctgaaag-3 '(SEQ ID NO: 3) and 5'-ccgtttcctagttcttccttgc-3' (SEQ ID NO: 4) using this cDNA as a template. went. The obtained PCR product was used for agarose electrophoresis to evaluate the expression of CHOP mRNA. GAPDH expression was used as a control as an internal standard. As a result, there was no dramatic suppression of CHOP expression induction observed in the TG stimulation in the group containing the constituent crude drugs other than TJ-54 and the addition group. The results are shown in FIG.

<Example 15>
The effect of Yokukansan on Alzheimer's disease-causing gene PS1 mutant cells that became vulnerable to ER stress was examined.
Human neuroblastoma SK-N-SH cells that are forced to express the mutant Presenilin-1delta exon 9 (PS1 △ E9) of familial Alzheimer's disease patients (FAD) (PS1 △ E9 is steadily expressed) Cells, also referred to as “mutant PS1ΔE9 expressing cells”), the effect of yokukansan on ER stress-induced cell death was examined. As a control, cells in which wild-type Presnilin-1 (PS1Wt) was forcibly expressed in human neuroblastoma SK-N-SH cells (cells that constantly express PS1Wt) were used. These mutant PS1ΔE9 expressing cells and control cells are both presenilin-1 mutation activates the signaling pathway of caspase-4 in endoplasmic reticulum stress-induced apoptosis: Yukioka F, et.al. Neurochem Int. 2008 Mar-Apr ; 52 (4-5): 683-7. The cell culture method and TG stimulation (addition) conditions were the same as in Example 11. The cell death assay was performed using an MTS assay kit (Promega). As a result, in the cells expressing the mutant PS1ΔE9 found in FAD patients, the percentage of surviving cells is 60% or more based on the ratio of surviving cells in PS1Wt expressing cells (control: normal cells) 3 hours after TG2 μM stimulation. Diminished. In contrast, when TJ-54 was added to the medium at a final concentration of 400 μg / mL 1.5 hours before TG loading, the ratio of the surviving cells recovered to about 80%. Thus, Yokukansan also showed an effect on cell death induced by PS1ΔE9 (N = 4). The results are shown in FIG. In FIG. 23, NT is a cell to which TG was not added, and TG is a cell to which TG was added (stimulated). Δ9 is the case where TJ-54 was not added to the mutant PS1ΔE9-expressing cells, and Δ9 + y is the case where TJ-54 (400 μg / mL in the medium) was added to the mutant PS1ΔE9-expressing cells.

<Example 16>
The effect of the constituent herbal medicines Shibahu and Kawakyu on Alzheimer's disease-causing gene PS1 mutant cells was examined.
In the same manner as in Example 15, human neuroblastoma SK-N-SH cells were forced to express a mutant Presenilin-1delta exon 9 (PS1ΔE9) possessed by a familial Alzheimer's disease patient (FAD) (PS1ΔE9) Using cells that constantly express E9, also referred to as “mutant PS1ΔE9-expressing cells”), the effect of TJ-54 on the ER stress-induced cell death of the constituent crude drugs Saiko and Kawakyu was examined. As in Example 15, as a control, cells in which wild-type Presnilin-1 (PS1Wt) was forcibly expressed in human neuroblastoma SK-N-SH cells (cells that constantly express PS1Wt) were used. Cell culture methods and TG stimulation (addition) conditions were the same as in Example 11. The cell death assay was performed using the MTS assay kit as in Example 15. As a result, in cells expressing the mutant PS1ΔE9 found in FAD patients, 2 to 200 μg / 200 μg / kg of cells were present on the basis of the ratio of viable cells in the group without TG stimulation (addition) (DMSO2) after 3 hours of TG2 μM stimulation. Pretreatment with mL suppressed the decrease of viable cells due to TG stimulation in a manner dependent on the amount of river cucumber added (FIG. 24, N = 4). On the other hand, there was a group in which the effect was seen in the pre-treatment group for Shiahu, but no dose dependency was seen (FIG. 25). 24 and 25, DMSO2 was a group that was not stimulated (added) by TG, and neither Kawakyu nor Shiba was added (control); TG2 was a group stimulated by TG 2 μM. In FIG. 24, TG2 + sen25 is a group stimulated with TG2 μM after a pretreatment of 25 μg / mL of river stream; TG2 + sen50 is a group stimulated with TG2 μM after a pretreatment of 50 μg / mL of river stream; TG2 + sen100 is a group of 100 μg / mL of river stream TG2 + sen200 is a group stimulated with TG2 μM after pretreatment with 200 μg / mL of river cucumber. In FIG. 25, TG2 + sai25 is a group stimulated with TG2 μM after pretreatment with 25 μg / mL of shihu; TG2 + sai50 is a group stimulated with 50 μg / mL of shihu and TG2 μM; TG2 + sai100 is 100 μg / mL of shigahu TG2 + sai200 is a group stimulated with TG2 μM after pretreatment with 200 μg / mL of shihu.

<Example 17>
The effects of Kawakyu and Yokukansan on GRP78 expression were examined.
Either Yokukansan or Kawakyu was added to the human neuroblastoma SK-N-SH cell culture solution so that the final concentration was 200 μg / mL (the control cells include Yokukansan and Kawakyu). None of the cucumbers were added). Each cell was recovered 6.5 hours after the addition, and RT-PCR was performed by the method used in Example 13. The GRP78 primer used and the PCR conditions were the same as those used in Example 13.
In SK-N-SH cells, the expression of GRP78 was significantly increased by both Yokukansan and Kawakyu. The results are shown in FIG.

<Example 18>
The effects of Kawakyu and Yokukansan on the expression of CHOP were examined.
Either Yokukansan or Kawakyu was added to the culture medium of human neuroblastoma SK-N-SH cells so that the final concentration was 200 μg / mL. TG 1 μM was added 1.5 hours after the addition. Neither Yokukansan nor Kawakyu was added to the control cells. After 4 hours, each cell was collected, and RT-PCR was performed by the method used in Example 14. The CHOP primer and PCR conditions used were the same as the CHOP primer and PCR conditions used in Example 14.
As a result, CHOP induction during TG loading was significantly suppressed by both pretreatment with Kawakyu and Yokukansan. The results are shown in FIG.

  As shown in the results of Examples 1 to 18, Yokukansan and Yokukansan, a constituent of Yokukansan, have significant inhibitory effects on ER stress-induced cell death and cell death promoting effects in Alzheimer's disease model cells. showed that. The increase in GRP78 expression and the effect of suppressing CHOP induction were involved in the effect of suppressing cell death by Yokukansan and Kawakyu. These results indicate that yokukansan increases the ER stress response and is useful not only for peripheral symptoms seen in neurodegenerative diseases and cerebral ischemia, but also for core symptoms.

  The present invention is useful for the prevention and treatment of neuronal cell death-related diseases caused by endoplasmic reticulum stress.

Claims (10)

  A preventive or therapeutic agent for neuronal cell death-related diseases caused by endoplasmic reticulum stress, comprising yokukansan.   The preventive or therapeutic agent according to claim 1, wherein the neuronal cell death-related disease caused by endoplasmic reticulum stress is Alzheimer's disease, Parkinson's disease, polyglutamine disease, mad cow disease, cerebral ischemia, or amyotrophic lateral sclerosis.   The preventive or therapeutic agent according to claim 1 or 2, wherein the neuronal cell death-related disease caused by endoplasmic reticulum stress is Alzheimer's disease or cerebral ischemia.   A prophylactic or therapeutic agent for neuronal cell death-related diseases caused by endoplasmic reticulum stress, comprising as an active ingredient at least one herbal medicine selected from the group consisting of river cucumber and saiko.   The preventive or therapeutic agent according to claim 4, wherein the crude drug is Kawakyu.   The prevention or treatment according to claim 4 or 5, wherein the neuronal cell death-related disease caused by endoplasmic reticulum stress is Alzheimer's disease, Parkinson's disease, polyglutamine disease, mad cow disease, cerebral ischemia, or amyotrophic lateral sclerosis. Agent.   The preventive or therapeutic agent according to claim 5 or 6, wherein the neuronal cell death-related disease caused by endoplasmic reticulum stress is Alzheimer's disease or cerebral ischemia.   An agent for preventing or suppressing neuronal cell death caused by endoplasmic reticulum stress, comprising yokukansan.   An agent for preventing or suppressing neuronal cell death due to endoplasmic reticulum stress, comprising as an active ingredient at least one herbal medicine selected from the group consisting of river cucumber and shiba hu.   A preventive or suppressive agent for endoplasmic reticulum stress or endoplasmic reticulum stress characterized by containing as an active ingredient at least one herbal medicine selected from the group consisting of Yokukansan or Kawakyu and Saiko.