4. 총명탕의 작용
알쯔하이머병(AD)의 원인에 대한 가설에는 뇌의 astrocyte와 microglial cell 등의 신경교세포에서 생성되는 proinflammatory cytokines에 의해 신경세포의 사멸이 촉진된다는 것이 있다. 이는 물리적 뇌손상, 감염 및 염증반응 등으로 인하여 Blood Brain Barrier가 손상되면 면역계 세포들이 중추신경계로 침입하여, 활성화된 astrocyte 및 microglia cell이 IL-1, IL-6, TNF 등의 proinflammatory cytokines들을 과다하게 분비하여 뇌에서의 cytokine 항상성이 파괴되고, 중추신경계에 염증반응이 일어난다는 가설로, 최근 활발한 연구가 이루어지고 있다.
정상상태의 뇌에서 astrocyte 및 microglial cell은 신경세포의 분화에 필요한 영양인자로서, cytokine들을 미량 분비함으로써 뇌의 생체 항상성을 유지하는데 기여하지만, 뇌손상에서 제일 먼저 병변 부위에 나타나는 microglial cell로 astrocyte의 주요자극인자인 IL-1 및 TNF-α를 생산하여 astrocyte를 활성화시키고 , 자극된 astrocyte는 IL-6, IL-8, TNF-α, GM-CSF, IL-5 등의 cytokine을 생성시키고 활성산소의 일종인 peroxynitrite를 유도함으로써 신경세포의 사멸을 야기하게 된다 .
또한 AD, Parkinson's disease 등의 퇴행성 뇌질환 병변 부위에서 IL-1, IL-6, TNF-α 등의 cytokine 발현을 보이는 astrocyte와 microglial cell의 활성화가 관찰됨으로써, 이들 질병과 proinflammatory cytokines가 밀접한 관계가 있으리라는 가설에 관심이 집중되고 있다81). 특히 βA가 침적된 노인반 주변의 microglial cell은 βA에 의해 활성화되어 IL-1, TNF-α 등의 proinflammatory cytokines을 생성함으로써 AD의 병리기전에 기여하는 것으로 알려져 있다 .
聰明湯 및 山査聰明湯은 PC-12 세포주에서 APP, AChE 유전자의 발현과 AChE의 activity, APP의 발현을 억제하였으며, βA로 유도된 AD 병태 생쥐모델에 대해 Morris water maze를 통한 stop-through latency, distance movement-through latency 측정에서 유의성 있는 기억력 개선 효과를 나타냈고 또한, βA로 유도된 AD 병태 생쥐모델의 microglial cell에서 proinflammatory cytokines인 IL-1β, TNF-α의 발현을 억제하였고, 뇌조직 MDA 값의 유의성 있는 감소효과를 나타내었으며, 뇌조직의 허혈상태를 개선하는 것으로 나타났다.
또한 聰明湯 및 山査聰明湯의 효과를 비교하여 보면, PC-12 세포주 수준의 in vitro 연구에서는 큰 차이를 보이지는 않았으나, βA로 유도된 AD 병태 생쥐에 대해 기억력 감퇴 억제, microglial cell에서 IL-1β, TNF-α 발현 억제, MDA 감소, 허혈 크기 감소효과에서는 모두 山査聰明湯이 聰明湯에 비해 우수한 효과를 보였으며, 특히 microglial cell에서 IL-1β, TNF-α 발현 억제, MDA 감소, 허혈 크기 감소효과에서 山査聰明湯은 acetylcholine 분해억제제로 FDA의 승인을 받아 현재 사용되는 양성대조군인 tacrine과 유사한 효과를 나타내는 것으로 관찰되었다.
Effect of 총명탕(CMT) and 산사총명탕(SCMT) on the AChE Activity in CT-105 plus rIL-1b Stimulated PC-12 Cells
Rat pheochromocytoma cell line(PC-12) were pretreated with various concentration CMT and SCMT(100, 10mg/ml) in the presence CT-105(20mM) plus rIL-1b(100ng/ml) for 24 h. The lysate(100ml,㎎/ml) were measured using AChE diagnostic kits. The other methods for assay were performed as described in Materials and Methods.
Normal was not treated, CT-105 plus rIL-1b was control, CT-105 plus rIL-1b + CMT and SCMT(100mg/ml), and CT-105 plus rIL-1b + CMT and SCMT(10mg/ml). Statistically significant value compared with CT-105 plus rIL-1b control group data by T test(*p<0.05, p<0.01, *p<0.001).
Effect of CMT and SCMT on the AChE activity in CT-105 plus rIL-1b stimulated PC-12 cells.
Rat pheochromocytoma cell line(PC-12) were pretreated with various concentration CMT and SCMT(100, 10mg/ml) in the presence CT-105(20mM) plus rIL-1b(100ng/ml) for 24 h. The lysate(100ml,㎎/ml) were measured using AChE diagnostic kits. The other methods for assay were performed as described in Materials and Methods.
Normal was not treated, CT-105 plus rIL-1b was control, CT-105 plus rIL-1b + CMT and SCMT(100mg/ml), and CT-105 plus rIL-1b + CMT and SCMT(10mg/ml). Statistically significant value compared with CT-105 plus rIL-1b control group data by T test(*p<0.05, p<0.01, *p<0.001).
Suppression effect of CMT and SCMT on the APP in PC-12 stimulated CT-105 plus rIL-1b by Western blot analysis.
Rat pheochromocytoma cell line(PC-12) were pretreated with various concentration CMT and SCMT in the presence CT-105(20mM) plus rIL-1b(100ng/ml) for 24 h. APP expression was measured by immune Western blot assay. Cell lysate(50mg/lane) were separated by SDS-polyacryamide gel electrophoresis and then transferred PDVF membranes. RPMI-1640 media control was not treated(A), CT-105 20mM plus recombinent IL-1b 100ng/ml Control(B), CMT 100 mg/ml(C), CMT 10mg/ml(D), SCMT 100mg/ml(E), SCMT 10mg/ml(F).
Effect of CMT and SCMT on the bA-induced Alzheimer's Mice of Impairment of Learning and Memory in the Stop-through Type Morris Water Maze Test
C57BL/6 mice were orally administered of CMT and SCMT or Tacrine.HCl and the acquisition training trial continued once a day for 60 days. The Stop-VIDEOTRACKING was initiated at 4 week and 8 week after orally administered of CMT(216㎎/kg) and SCMT(576㎎/kg) the retention trial was carried out after day 7 after the acquisition training trial(pre-training) and injected bA-induced mice.
Control, bA-induced Alzheimer's mice; Tacrine(10㎎/kg p.o); CMT(216㎎/kg p.o); SCMT(576㎎/kg p.o). Stop-though latency each time the animal enters an area, the entries counter for this area is incremented by one. Statistically significant value compared with bA-induced Alzheimer's mice group(Control) data by T test(*p<0.05, p<0.01, *p<0.001).
Effects of CMT and SCMT on the bA-induced Alzheimer's mice of impairment of learning and memory in the stop-through type Morris water maze test.
C57BL/6 mice were orally administered of CMT and SCMT or Tacrine.HCl and the acquisition training trial continued once a day for 60 days. The Stop-VIDEOTRACKING was initiated at 4 week and 8 week after orally administered of CMT(216㎎/kg) and SCMT(576㎎/kg) the retention trial was carried out after day 7 after the acquisition training trial(pre-training) and injected bA-induced mice.
Control, bA-induced Alzheimer's mice; Tacrine(10㎎/kg p.o); CMT(216㎎/kg p.o); SCMT(576㎎/kg p.o). Stop-though latency each time the animal enters an area, the entries counter for this area is incremented by one. Statistically significant value compared with bA-induced Alzheimer's mice group(Control) data by T test(*p<0.05, p<0.01, *p<0.001).
Effect of CMT and SCMT on the IL-1b activity on the brain CD14+cells in bA-Alzheimer's disease mice model.
C57BL/6 mice were orally administered of CMT(216㎎/kg), SCMT(576㎎/kg) and tacrine(10㎎/kg) for 60 days. continued once a day for 60 days. IL-1b activity measured by FACScalibur., number of CD14 positive cells in the mouse brain of control and the groups were submitted during the stereotaxic procedures to bA-induced Alzheimer's disease model. Statistically significant value compared with bA-induced Alzheimer's mice group(Control) data by T test(*p<0.05, p<0.01, *p<0.001).
Effect of CMT and SCMT on the TNF-a activity on the brain CD14+cells in bA-Alzheimer's disease mice model.
C57BL/6 mice were orally administered of CMT(216㎎/kg), SCMT(576㎎/kg) and tacrine(10㎎/kg) for 60 days. continued once a day for 60 days. TNF-a activity measured by FACScalibur., number of CD14 positive cells in the mouse brain of control and the groups were submitted during the stereotaxic procedures to bA-induced Alzheimer's disease model. Statistically significant value compared with bA-induced Alzheimer's mice group(Control) data by T test(*p<0.05, p<0.01, *p<0.001).(하수영(2004)의 논문에서 발췌)
