JP5572845B2 - Composition for inhibiting activation of glial cells - Google Patents

Description

  The present invention relates to a composition for suppressing activation of glial cells and the like.

  A bush (aconiti tuber) is a herbal medicine extracted from the tuberous root of a plant belonging to the genus Aconitium, and is known to have a cardiotonic action and an analgesic action. However, the bushes are highly toxic as they are, and so treatment (heating / pressurization) is usually performed to attenuate them. Toxic substances such as aconitine and mesaconitine contained in bushes are changed into benzoylaconine and benzoylmesaconine, respectively, after treatment.

  A glial cell (glial cell) is a general term for cells that are not neurons in the central nervous system, and includes astrocytes, microglia, oligodendrocytes, upper garment cells, Schwann cells, satellite cells, etc. . Until now, glial cells were thought not to be involved in signal transduction, so in the study of the central nervous system, the main focus was on elucidating the mechanisms of nerve cells, and the role of glial cells was mostly studied. It was not progressing. However, in recent years, research on glial cells has gradually progressed, and various roles of glial cells have been clarified. The role of glial cells is, for example, to fix the position of nerve cells, to synthesize and secrete neurotrophic factors, to be a component of myelin, and to re-uptake ions such as potassium released excessively. Functions to maintain the homeostasis of the surrounding tissues of nerve cells, such as limiting the transmission time by collecting neurotransmitters in the cell, forming a blood brain barrier with the vascular endothelium, and acting as a filter Besides, it has been known to express receptors for various neurotransmitters and contribute to more aggressive signal transduction.

  An example of a signaling substance for signal transduction involving glial cells is ATP. When a nerve cell is damaged, a large amount of ATP is released from the nerve cell, and microglia that senses this ATP migrates to the damaged site, produces various humoral factors, and contributes to the repair of nerve function. However, when it cannot be repaired, microglia exert phagocytosis and remove dead cells.

  On the other hand, glial cells are also known to be excessively activated in, for example, spinal cord trauma, neuropathy due to stroke, epilepsy, neuropathic pain, vaso-occlusive eye disease, neurodegenerative disease, etc. . When glial cells are excessively activated, various injury factors such as glutamic acid, nitric oxide, and inflammatory cytokines are produced, and the function of nerve cells is impaired (Non-Patent Documents 1 to 3). Therefore, excessive activation of glial cells is considered to be the cause of the above-mentioned trauma and disease and a factor of exacerbation.

  However, a practical drug capable of suppressing excessive activation of glial cells has not been known so far.

Benvenisite, Am J Physiology, 1992, 263, 1-16 Fields, et al., Science, 2002, 298, 556-562 Suzumura et al., Ann. N.Y. Acad. Sci., 2006, 1088, 219-229

  An object of the present invention is to provide a glial cell activation inhibitory composition and the like that are capable of suppressing excessive activation of glial cells and that are practical.

  The present inventor has intensively studied to solve the above-mentioned problems, and the healing bushi did not suppress the activation of glial cells during the pain formation period and did not exhibit the analgesic effect, whereas the pain maintenance period The inventors found that the activation of glial cells was suppressed and the analgesic effect was exerted, and the present invention was completed based on this finding.

That is, the present invention is, glial for administration to a subject having a disease selected from the group consisting of (1) Te epilepsy, vascular occlusive eye disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS) A composition for inhibiting the activation of cells, comprising the composition for inhibiting the activation of glial cells containing sushi bushi, and (2) the glial cells are microglia or astrocytes The composition for inhibiting the activation of glial cells according to (2) above, wherein (3) the activation of astrocytes is an activation accompanied by an increase in the expression level of pERK. A composition for suppressing activation of glial cells according to (2) above, wherein (4) the inhibition of microglial activation is a secondary change due to deactivation of astrocytes, or (5) do Te Vascular occlusive eye diseases, in the preparation of Alzheimer's disease and amyotrophic lateral sclerosis activator inhibitor glial cells for administration to a subject having a disease selected from the group consisting of (ALS), Shuji sieboldiana About the use of.

The invention also relates to (6) Shuji containing busi, a therapeutic agent for diseases activation of glial cells are involved, epilepsy Te said disease, vascular occlusive eye disease, Alzheimer's disease and muscle is a disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), and therapeutic agents, (7) diseases is Alzheimer's disease or amyotrophic lateral sclerosis is a (ALS) above (6) It relates to the therapeutic agent described in 1.

  According to the present invention, excessive activation of glial cells can be effectively suppressed. Therefore, the activation inhibitory composition for glial cells in the present invention can also be used for prevention and treatment of diseases caused by excessive activation of glial cells. Moreover, since the Shuji Bushi in this invention is already used as a Chinese medicine, the glial cell activation suppression composition in this invention has high safety | security to a human body.

It is a figure which shows the result of the analgesic effect test 1 using the neuropathic pain mouse | mouth. The left figure shows the result of thermal hyperalgesia reaction test, and the right figure shows the result of von Frey test. In both the left and right diagrams, the graphs for each period represent the Sham-Water group, the Ligation-Water group, and the Ligation-Bushimatsu group from the left. It is a figure which shows the result of the analgesic effect test 2 using the neuropathic pain mouse | mouth. The left figure shows the result of thermal hyperalgesia reaction test, and the right figure shows the result of von Frey test. In both the left and right diagrams, the graphs for each period represent the Sham-Water group, the Ligation-Water group, and the Ligation-Bushimatsu group from the left. It is a figure which shows the result of the morphology observation 1 of the spinal cord microglia. It is a figure which shows the result of the morphological observation of the intra-spinal astrocyte. It is a figure which shows the result of the morphology observation 2 of the microglia in a spinal cord. It is a figure which shows the result of the expression assay of pERK in a spinal cord astrocyte. It is a figure which shows the result of the analysis of pERK in the spinal cord origin culture | cultivation astrocyte. The left figure shows the results of Western blotting for ERK1, ERK2, pERK1, and pERK2. The right figure shows the results of immunofluorescence staining of pERK localization using PI (propidium iodide) and anti-pERK antibody. It is. It is a figure which shows the test result at the time of administering an astrocyte specific metabolic inhibitor to a neuropathic pain mouse | mouth. The left figure shows the results of the analgesic effect test (von Frey test), and the right figure shows the results of GFAP staining. In addition, the graph of each time in the left figure represents the graph of Sham-Veh group, Ligation-Veh group, and Ligation-FC group from the left. It is a figure which shows the test result at the time of administering an astrocyte specific metabolic inhibitor to a neuropathic pain mouse | mouth. The left figure shows the results of the analgesic effect test (von Frey test), and the right figure shows the results of GFAP staining and pERK staining. In addition, the graph of each time in the left figure represents the graph of Sham-Veh group, Ligation-Veh group, and Ligation-FC group from the left.

  The composition for inhibiting activation of glial cells according to the present invention is not particularly limited as long as it contains a healing bush. As used herein, the term “shushibushi” refers to those obtained by attenuating tuberous roots of plants belonging to the genus Ranunculaceae. Specifically, for 1 g of dried product, aconitine is 55 μg or less, jessaconitine is 40 μg or less, and hipaconitine is contained. 55 μg or less, and mesaconitin is 120 μg or less, and further includes Bushi (refer to the second supplement of Japanese Pharmacopoeia, December 14, 2004), wherein the total weight of these four kinds of toxic components is 230 μg or less. . The attenuated treatment is not particularly limited as long as the four kinds of toxic components contained in the bush fall below the above-mentioned standard due to the attenuated treatment, for example, a method of treating the bush with high-pressure steam, Examples thereof include a method in which a bush is immersed in a saline solution and then treated with high-pressure steam, and a method in which lime is applied after the bush is immersed in a saline solution.

  Examples of the plant belonging to the genus Ranunculaceae include, butterflyfish, octopus butterfly, chives, azulfish beetle, calacon beetle, chii sanbutsubu, kibana akite, ibuki akite, camellia beetle, yamato akite, etc. Among these, the flowering beetle and the cocklefish can be preferably exemplified.

  As described above, the healing bush used in the present invention may be produced by attenuating a tuberous root of a plant belonging to the genus Ranunculaceae, or using a commercially available healing bush (also referred to as “processed bush”). Good. Shuji Bushi is widely marketed as a powdered processed bush.

  The glial cell in the glial cell activation-suppressing composition of the present invention means a cell that is not a nerve cell among cells constituting the central nervous system. For example, astrocytes, microglia, oligodendrocytes, upper garments Examples thereof include cells, Schwann cells, satellite cells, etc. Among them, microglia and astrocytes can be preferably exemplified.

  The glial cell activation inhibitory effect of the glial cell activation inhibitory composition of the present invention means an effect of suppressing excessive glial cell activation, for example, accompanied by an increase in the expression level of astrocyte pERK It preferably includes the effect of suppressing excessive activation and the effect of suppressing microglia activation, which is a secondary change caused by deactivation of astrocytes. Whether the glial cells are excessively activated or has an effect of suppressing the excessive activation of glial cells is determined by collecting spinal cord tissue from non-human animals, etc. This can be confirmed by immunofluorescent staining of the tissue section described above using an antibody that specifically binds to cells. For example, if the signal of the glial cell in a non-human animal is stronger than the signal of the glial cell in a normal non-human animal, the glial cell is excessively activated in the non-human animal Can be evaluated. In addition, when a certain substance is administered to a non-human animal in which glial cells are in an excessively activated state and the signal of the glial cells decreases, the substance suppresses excessive activation of glial cells. It can be evaluated that it has an effect. The glial cell activation-suppressing composition of the present invention can be added to and blended with foods and supplements.

  The glial cell activation-suppressing composition of the present invention may contain only the healing bushi, but may additionally contain excipients, antioxidants, stabilizers, other analgesics and the like. . The content of the healing bushi contained in the glial cell activation-suppressing composition of the present invention is not particularly limited, but is 0.01% by mass to 100% by mass, preferably 10% by mass to the total amount of the composition. The amount may be 100% by mass, more preferably 50% by mass to 100% by mass.

  The dosage form of the glial cell activation-suppressing composition of the present invention is not particularly limited, but examples include solids such as powders, granules, fine granules, tablets, capsules, liquids, and syrups. From the viewpoint of storage stability, a solid agent can be preferably exemplified.

  The composition for inhibiting activation of glial cells of the present invention may be prepared by using Shuji Bushi as it is, or may be prepared by mixing ingredients according to the above-mentioned dosage form in addition to Shuji Bushi. Good.

  As the administration form of the glial cell activation-suppressing composition of the present invention, oral administration can be preferably exemplified. The dose is not particularly limited as long as the glial cell activation inhibitory effect of the present invention can be obtained, but can be exemplified within the range of 10 mg / kg to 5000 mg / kg per day, and 50 mg / kg per day. A range of ˜1000 mg / kg can be preferably exemplified.

  Examples of the use in the present invention include the use of Shuji Bushi in the preparation of pERK inhibitors, pERK nuclear translocation inhibitors, or glial cell activation inhibitors. The pERK inhibitor means an agent that suppresses phosphorylation of ERK, and the pERK nuclear translocation inhibitor means an agent that inhibits pERK from translocating into the nucleus of a cell.

  The therapeutic agent for a disease involving activation of glial cells of the present invention is not particularly limited as long as it contains a healing bushi. The disease involving the activation of glial cells is not particularly limited as long as it is a disease caused by excessive activation of glial cells or a disease whose symptoms are worsened by excessive activation of glial cells. The disease selected from the group consisting of neuropathy caused by stroke, epilepsy, neuropathic pain, vaso-occlusive eye disease, neurodegenerative disease can be preferably exemplified, and as the above-mentioned neurodegenerative disease, Alzheimer's disease Amyotrophic lateral sclerosis (ALS) can be illustrated more suitably. Since Shuji Bushi can suppress excessive activation of glial cells, the therapeutic agent of the present invention containing Shuji Bushi has a prophylactic effect or a therapeutic effect on diseases involving the activation of these glial cells. Can be demonstrated. The content, dosage form, preparation method, dosage form, dosage, and the like of the therapeutic bushi in the therapeutic agent of the present invention can be the same as those in the glial cell activation-suppressing composition described above.

[Preparation of neuropathic pain model mice]
An attempt was made to create a Seltzer model mouse as a neuropathic pain model mouse. Specifically, the mouse was prepared by exposing the sciatic nerve on one leg of a mouse and ligating 30-50% of the sciatic nerve with 6-0 silk thread. In this model mouse, pain gradually progresses from 1 week to 10 days after ligation surgery (pain formation phase), and then chronic pain persists (pain maintenance phase).

[Analgesic effect test 1]
The following analgesic effect test was performed using the neuropathic pain mouse prepared in Example 1. First, the schedule of the analgesic effect test is shown in “Schedule” of FIG. Immediately after the sciatic nerve ligation operation, 0 day is set, and immediately before the operation (Pre), 1 day after the operation (D1), 3 days (D3), 7 days (D7), 14 days (D14) ) After 21 days (D21), two types of analgesic effect tests, a thermal hyperalgesia reaction test and a von Frey test, were performed. At that time, three groups of mice (a) to (c) were used. That is, (a) a group in which water was orally administered to non-ligated mice (Sham-Water group), (b) a group in which water was orally administered to neuropathic pain mice prepared in Example 1 (Ligation-Water group), (C) Three groups of the group (Ligation-Bushimatsu group) in which the neuropathic pain mice prepared in Example 1 were treated with 300 mg / kg of Shuji Bushi once a day for chronic oral administration immediately after ligation surgery. In addition, Shuji Bushi in all subsequent experiments used what was purchased from Tsumura Corporation.

(1) Thermal hyperalgesia test The thermal hyperalgesia test was performed according to a conventional method. That is, a projection thermal stimulus was applied to the sole of the mouse, and the latency of escape reflex (withdrawal latency (sec.)) Was measured. The smaller the value of withdrawal latency, the higher the sensitivity to heat.

(2) von Frey test
The von Frey test was performed according to a conventional method. That is, the mouse was placed on a net, pressed from the bottom of the net until the filament (0.166 g) was bent perpendicularly to the sole of the mouse, and the threshold of mechanical stimulation by which the mouse lifted the foot was measured. The larger the “% of max score” value, the higher the sensitivity to pain.

  The results of the thermal hyperalgesia test and the von Frey test are shown in the graph of “Thermal hyperalgesia” and the graph of “von Frey test (0.166 g)” in FIG. The graphs of each period in both graphs represent graphs of the Sham-Water group, the Ligation-Water group, and the Ligation-Bushimatsu group from the left. As can be seen from the graph of “Thermal hyperalgesia” in FIG. 1, there was no significant difference in “withdrawal latency” between the Ligation-Water group and the Ligation-Bushimatsu group during the pain formation period up to D7. In the pain maintenance period such as D14 and D21, the “withdrawal latency” of the Ligation-Bushimatsu group was significantly higher than the “withdrawal latency” of the Ligation-Water group. In addition, as can be seen from the graph of “von Frey test (0.166 g)” in FIG. 1, there is no significant difference in “% of max score” between the Ligation-Water group and the Ligation-Bushimatsu group until D3. However, in D7, D14 and D21, the “% of max score” in the Ligation-Bushimatsu group was significantly lower than the “% of max score” in the Ligation-Water group. Based on these facts, Shuji Bushi does not exert much analgesic effect on pain in the period of pain formation up to about D7, and exerts remarkable analgesic effect on pain in the subsequent pain maintenance period. It has been shown.

[Analgesic effect test 2]
The following analgesic effect test was performed using the neuropathic pain mouse prepared in Example 1. First, the schedule of the analgesic effect test is shown in “Schedule” of FIG. This analgesic effect test was conducted in the Example except that the treatment time of Shuji Bushi in the Ligation-Bushimatsu group was limited to the period of 7 to 21 days after the sciatic nerve ligation operation instead of every day immediately after the sciatic nerve ligation operation. The analgesic effect test 1 was conducted in the same manner as in 2.

  The results of the thermal hyperalgesia test and the von Frey test are shown in the “Thermal hyperalgesia” graph and the “von Frey test (0.166 g)” graph of FIG. 2, respectively. The graphs of each period in both graphs represent graphs of the Sham-Water group, the Ligation-Water group, and the Ligation-Bushimatsu group from the left. As can be seen from the graph of “Thermal hyperalgesia” in FIG. 2, there was no significant difference in “withdrawal latency” between the Ligation-Water group and the Ligation-Bushimatsu group during the pain formation period up to D7. In the subsequent pain maintenance period, the “withdrawal latency” of the Ligation-Bushimatsu group was almost significantly higher than the “withdrawal latency” of the Ligation-Water group. As can be seen from the graph of “von Frey test (0.166 g)” in FIG. 2, there is no significant difference in “% of max score” between the Ligation-Water group and the Ligation-Bushimatsu group until D7. After that, the “% of max score” in the Ligation-Bushimatsu group was significantly lower than the “% of max score” in the Ligation-Water group. From these results, it was shown that Shuji Bushi exerts a remarkable analgesic effect even when chronically administered after pain has been formed (after D7).

[Form observation of microglia in spinal cord 1]
In order to examine the effect of Shuji Bushi on spinal cord glial cells, the morphology of spinal cord microglia was observed as follows. First, the schedule for morphological observation of spinal cord microglia is shown in “Schedule” in FIG. In the Ligation-Bushimatsu group in this morphological observation, Shuji Bushi was administered chronically from D7 to D21.

  Spinal cords were collected at D10, D14, and D21 for three groups of Sham-Water group, Ligation-Water group, and Ligation-Bushimatsu group, respectively, and paraffin-embedded tissue sections were prepared for the dorsal horn of the spinal cord. Each tissue section was immunofluorescently stained using an anti-Iba1 antibody that specifically binds to a microglia-specific calcium binding protein Iba1 (Ionized calcium binding adapter molecule 1). The result is shown in FIG. As can be seen from the results of the Ligation-Water group, the enhancement of the Iba1 signal continues from D10 to D21, and the activation of microglia continues. On the other hand, in the Ligation-Bushimatsu group, the Iba1 signal gradually decreased from D10 to D21, and the healing bushi suppresses the activation of microglia during the pain maintenance period (from activated microglia to stationary microglia). Change).

[Morphological observation of spinal astrocytes]
In order to investigate the effect of Shuji Bushi on spinal cord glial cells, the morphology of spinal cord astrocytes was observed as follows. First, the schedule for morphological observation of spinal cord astrocytes is shown in “Schedule” in FIG. In the Ligation-Bushimatsu group in this morphological observation, chronic administration of Shuji Bushi was performed from D7 to D21 as in the experiment of Example 4.

  Spinal cords were collected at D10, D14, and D21 for three groups of Sham-Water group, Ligation-Water group, and Ligation-Bushimatsu group, respectively, and paraffin-embedded tissue sections were prepared for the dorsal horn of the spinal cord. Each tissue section was immunofluorescently stained using an anti-GFAP antibody that specifically binds to glial fibrillary acidic protein (GFAP). The result is shown in FIG. As can be seen from the results of the Ligation-Water group, the enhancement of the GFAP signal continues from D10 to D21, and the activation of astrocytes continues. On the other hand, in the Ligation-Bushimatsu group, the GFAP signal gradually decreased from D10 to D21, and it was shown that Shuji Bushi suppressed the activation of astrocytes in the dorsal horn of the spinal cord during the pain maintenance period. .

[Form observation of microglia in spinal cord 2]
In the above-mentioned Example 4, it was shown that the activation of microglia in the pain maintenance phase is suppressed by the healing sushi. In order to confirm whether this microglial activation suppression is also observed in the pain formation stage, the morphology of the microglia in the spinal cord was observed as follows. The schedule for morphological observation of spinal microglia this time is shown in “Schedule” in FIG. In the Ligation-Bushimatsu group in this form observation, Shuji Bushi was chronically administered from D1 to D7.

  Spinal cords were collected at D7 for each of the three groups of Sham-Water, Ligation-Water, and Ligation-Bushimatsu groups, and paraffin-embedded tissue sections were prepared for the dorsal horn of the spinal cord. Each tissue section was immunofluorescently stained using an anti-Iba1 antibody that specifically binds to a microglia-specific calcium binding protein Iba1 (Ionized calcium binding adapter molecule 1). The result is shown in FIG. As can be seen from FIG. 5, at the time point D7, which is the pain formation period, the activation of microglia in the dorsal horn of the spinal cord was not suppressed by the treatment with Shuji Bushi. That is, it was shown that the activation of microglia caused by sciatic nerve ligation was not suppressed even after chronic administration of Shuji Bushi.

[Expression assay of pERK in spinal cord astrocytes]
In astrocytes activated in the dorsal horn of the spinal cord by sciatic nerve ligation, it is known that MAPKinase (ERK) phosphorylation increases, that is, the expression level of pERK increases (Kawasaki et al., Nature medicine). , 2008, 14, p331-336). Therefore, it was decided to examine what kind of effect the administration of Shuji Bushi has on the expression of pERK in the astrocytes in the spinal cord. In the Ligation-Bushimatsu group in the pERK expression assay, chronic administration of Shuji Bushi was continued from D7 to D21.

  Spinal cords were collected at D21 for three groups of Sham-Water group, Ligation-Water group, and Ligation-Bushimatsu group, and paraffin-embedded tissue sections were prepared for the dorsal horn of the spinal cord. Each tissue section was immunofluorescently stained using an anti-GFAP antibody (labeled with green fluorescence) and an anti-pERK antibody (labeled with red fluorescence). The result is shown in FIG. As can be seen from FIG. 6, in the Ligation-Bushimatsu group, both the GFAP signal and the pERK signal are significantly suppressed as compared with the Ligation-Water group. From these results, it was shown that the activation of astrocytes accompanied by an increase in the expression level of pERK was significantly suppressed by chronic administration to Shuji Bushi.

[Analysis of pERK in spinal cord-derived cultured astrocytes]
In order to examine whether the ERK phosphorylation inhibitory effect exhibited by Shuji Bushi is a direct action on Shuji Bushi's astrocytes, we confirmed the effects of Shuji Bushi using spinal cord-derived cultured astrocytes did. The schedule of this test is shown in “Schedule” of FIG. Primary cultured astrocytes obtained by collecting cells from the spinal cord of mice were starved for 24 hours, and then incubated for 1 hour and 45 minutes in DMEM to which 100 μg / mL was added, and then IL- After incubation for 15 minutes in DMEM to which 1β was added at 10 ng / mL, astrocytes were sampled (Bushimatsu-IL-1β group). As controls, those in which only IL-1β was not added in the above-described method (Busimatsu-DMEM group), those in which only Shuji Bushi was not added in the above-described method (IL-1β group), and Shuji-Bushi in the above-described method. Sampling was also performed for those to which no IL-1β was added (DMEM group). IL-1β is known as an activator of astrocytes.

  Western blotting was performed on the proteins extracted from each sampled astrocyte, and the expression levels of ERK1, ERK2, pERK1, and pERK2 were detected. The result is shown in the left figure of FIG. The left bar graph in each group shows the percentage (%) when the expression level of pERK1 in the DMEM group is 100%, and the right bar graph shows the expression level of pERK2 in the DMEM group as 100%. The percentage of time is shown. From the result of the left figure of FIG. 7, it was shown that pERK which is increased when a cultured astrocyte is activated with IL-1β is suppressed by Shuji Bushi.

  IL-1β is known to enhance nuclear translocation of pERK in astrocytes. Therefore, in order to examine the effect of Shuji Bushi on the nuclear translocation of pERK, the localization of pERK in cultured astrocytes sampled according to the schedule shown in FIG. 7 was determined by using PI (propidium iodide) and anti-pERK antibody. It confirmed using. In the astrocytes of each of the aforementioned four groups (DMEM group, IL-1β group, Busimatsu-DMEM group, and Bushimatsu-IL-1β group), the ratio of astrocytes that were able to confirm the localization of pERK in the nucleus was measured. The results are shown in the right figure of FIG. From the result of the right figure of FIG. 7, it was shown that the nuclear translocation of pERK activated by IL-1β is suppressed by Shuji Bushi.

[Analgesic effect test and morphological observation of spinal astrocytes when an astrocyte-specific metabolic inhibitor is administered]
In order to confirm that the analgesic effect of Shuji Bushi administration was due to suppression of activation of astrocytes in the dorsal horn of the spinal cord, the following supplementary experiment was conducted. The schedule of this analgesic effect test is shown in “Schedule” of FIG. Fluorocitrate (FC), which is known as an astrocyte-specific metabolic inhibitor, was applied to the neuropathic pain model mouse prepared in Example 1 at 100 fmol / day from day 14 to day 28 after ligation surgery. 5 μL was administered chronically in the spinal cord. The result of the von Frey test on the model mouse is shown in the left diagram of FIG. The graph of each period in the graph of this figure represents the graph of Sham-Veh group, Ligation-Veh group, and Ligation-FC group from the left. Further, the results of morphological observation (GFAP staining) of spinal cord astrocytes on day 21 (D21) are shown in the right diagram of FIG. As can be seen from these figures, FC provided a remarkable analgesic effect in the pain maintenance phase, and the activation of astrocytes was significantly suppressed in the dorsal horn of the spinal cord at that time (D21).

  Further, for the mice in the above three groups (Sham-Veh group, Ligation-Veh group, Ligation-FC group), a paraffin-embedded tissue section in the dorsal horn of the spinal cord was prepared, and an anti-GFAP antibody (labeled with green fluorescence) FIG. 9 shows the result of immunofluorescence staining using a pERK antibody (labeled with red fluorescence). As can be seen from FIG. 9, in the Ligation-FC group (SNL-Fluorocitrate group), both the GFAP signal and the pERK signal are significantly suppressed as compared to the Ligation-Veh group (SNL-Vehicle group). From these results, it was shown that the activation of astrocytes accompanied by an increase in the expression level of pERK was significantly suppressed by chronic administration of FC into the spinal cord. Thus, the administration of FC resulted in the same results as the administration of Shuji Bushi. Therefore, the reduction and treatment effect of neuropathic pain by Shuji Bushi suppressed the activation of astrocytes in the dorsal horn of spinal cord (deactivation) It was shown that it is caused by

The present invention can be suitably used in fields related to glial cell activation-suppressing compositions.

Claims (7)

There Te epilepsy, vascular occlusive eye diseases, in the activation inhibiting composition glial cells for administration to a subject having a disease selected from the group consisting of Alzheimer's disease and amyotrophic lateral sclerosis (ALS) The composition which suppresses the activation of glial cells containing Shuji Bushi. The composition for inhibiting activation of glial cells according to claim 1, wherein the glial cells are microglia or astrocytes. The composition for inhibiting activation of glial cells according to claim 2, wherein the activation of astrocytes is an activation accompanied by an increase in the expression level of pERK. The composition for inhibiting activation of glial cells according to claim 2, wherein the inhibition of microglial activation is a secondary change caused by deactivation of astrocytes. Te epilepsy, vascular occlusive eye diseases, in the preparation of Alzheimer's disease and amyotrophic lateral sclerosis activator inhibitor glial cells for administration to a subject having a disease selected from the group consisting of (ALS) The use of Shuji Bushi. Containing Shuji busi, a therapeutic agent for diseases activation of glial cells are involved, it said disease, Te epilepsy, vascular occlusive eye disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS) A therapeutic agent, which is a disease selected from the group consisting of. Diseases is a therapeutic agent of claim 6 is Alzheimer's disease or amyotrophic lateral sclerosis (ALS).

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