Amyloid β에 의해 유도된 신경세포 손상에 대한 phosphodiesterase III inhibitor인 cilostazol의 신경보호 효과 Phosphodiesterase III Inhibitor Cilostazol Protects Amyloid β-Induced Neuronal Cell Injury via Peroxisome Proliferator-Activated Receptor-γ Activation원문보기
Amyloid ${\beta}$ ($A{\beta}$)의 신경독성은 알츠하이머병의 주된 원인이 되고 이러한 신경독성은 일련의 신경세포 사멸반응에 의해 일어난다고 알려져 있다. 본 연구에서는 알츠하이머병의 실험모델로 mouse primary neuronal cell에 $A{\beta}_{25-35}$를 처리하여 세포독성을 유도하는 세포실험모델과 C57BL/6J mouse 뇌실에 $A{\beta}_{25-35}$를 주입하여 인지장애를 일으키는 동물실험모델을 이용하여 phosphodiesterase III 억제제인 cilostazol의 신경보호 효과에 대해 조사하였다. $A{\beta}_{25-35}$를 신경세포에 처리하면 세포생존율이 감소되었고, 세포사멸이 일어난 세포의 수도 증가되었다. 이러한 $A{\beta}_{25-35}$에 의한 세포독성이 cilostazol처리에 의해 회복되었으며, peroxisome proliferator-activated receptor(PPAR)-${\gamma}$ 항진제인 rosiglitazone 또한 동일한 회복효과를 나타내었다. Cilostazol과 rosiglitazone에 의한 이러한 회복효과가 PPAR-${\gamma}$ 길항제인 GW9662에 의해 다시 억제되는 결과를 통해 cilostazol의 효과는 PPAR-${\gamma}$가 매개하는 신호전달이 관여함을 알 수 있었다. 직접 PPAR-${\gamma}$ 활성화 정도를 측정한 결과, $A{\beta}_{25-35}$ 처리에 의해 감소된 PPAR-${\gamma}$ 활성화 정도가 cilostazol과 rosiglitazone에 의해 증가함을 관찰할 수 있었고, 이는 GW9662에 의해 다시 억제됨을 확인하였다. 게다가, cilostazol은 세포사멸이 일어난 세포의 수와 세포사멸 조절단백질인 Bax/Bcl-2의 비율도 감소시켰다. Cilostazol (20 mg/kg, 구강투여)을 C57BL/6J mice 뇌실에 $A{\beta}_{25-35}$를 주입하기 2주 동안 전처리하고, $A{\beta}_{25-35}$ 주입 후 4주 동안 처리하면, 기억력과 학습능력을 증진시킨다는 결과를 water maze 실험을 통해 알 수 있었으며, rosiglitazone (10 mg/kg)을 먹인 동물에서도 동일한 결과를 얻을 수 있었다. 본 연구를 통해서 cilostazol이 PPAR-${\gamma}$ 활성화를 통해 $A{\beta}_{25-35}$로 인한 신경세포 손상과 세포사멸을 약화시켜, 신경세포의 생존을 증진시키고, 알츠하이머에서 인지장애를 개선할 것으로 생각된다. 따라서, phosphodiesterase III 억제제인 cilostazol은 알츠하이머 질병 치료에 새로운 전략이 될 수 있을 것이다.
Amyloid ${\beta}$ ($A{\beta}$)의 신경독성은 알츠하이머병의 주된 원인이 되고 이러한 신경독성은 일련의 신경세포 사멸반응에 의해 일어난다고 알려져 있다. 본 연구에서는 알츠하이머병의 실험모델로 mouse primary neuronal cell에 $A{\beta}_{25-35}$를 처리하여 세포독성을 유도하는 세포실험모델과 C57BL/6J mouse 뇌실에 $A{\beta}_{25-35}$를 주입하여 인지장애를 일으키는 동물실험모델을 이용하여 phosphodiesterase III 억제제인 cilostazol의 신경보호 효과에 대해 조사하였다. $A{\beta}_{25-35}$를 신경세포에 처리하면 세포생존율이 감소되었고, 세포사멸이 일어난 세포의 수도 증가되었다. 이러한 $A{\beta}_{25-35}$에 의한 세포독성이 cilostazol처리에 의해 회복되었으며, peroxisome proliferator-activated receptor(PPAR)-${\gamma}$ 항진제인 rosiglitazone 또한 동일한 회복효과를 나타내었다. Cilostazol과 rosiglitazone에 의한 이러한 회복효과가 PPAR-${\gamma}$ 길항제인 GW9662에 의해 다시 억제되는 결과를 통해 cilostazol의 효과는 PPAR-${\gamma}$가 매개하는 신호전달이 관여함을 알 수 있었다. 직접 PPAR-${\gamma}$ 활성화 정도를 측정한 결과, $A{\beta}_{25-35}$ 처리에 의해 감소된 PPAR-${\gamma}$ 활성화 정도가 cilostazol과 rosiglitazone에 의해 증가함을 관찰할 수 있었고, 이는 GW9662에 의해 다시 억제됨을 확인하였다. 게다가, cilostazol은 세포사멸이 일어난 세포의 수와 세포사멸 조절단백질인 Bax/Bcl-2의 비율도 감소시켰다. Cilostazol (20 mg/kg, 구강투여)을 C57BL/6J mice 뇌실에 $A{\beta}_{25-35}$를 주입하기 2주 동안 전처리하고, $A{\beta}_{25-35}$ 주입 후 4주 동안 처리하면, 기억력과 학습능력을 증진시킨다는 결과를 water maze 실험을 통해 알 수 있었으며, rosiglitazone (10 mg/kg)을 먹인 동물에서도 동일한 결과를 얻을 수 있었다. 본 연구를 통해서 cilostazol이 PPAR-${\gamma}$ 활성화를 통해 $A{\beta}_{25-35}$로 인한 신경세포 손상과 세포사멸을 약화시켜, 신경세포의 생존을 증진시키고, 알츠하이머에서 인지장애를 개선할 것으로 생각된다. 따라서, phosphodiesterase III 억제제인 cilostazol은 알츠하이머 질병 치료에 새로운 전략이 될 수 있을 것이다.
The neurotoxicity of aggregated amyloid ${\beta}$ ($A{\beta}$) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD). It can cause neurotoxicity in AD by evoking a cascade of apoptosis to neuron. Here, we investigated the neuroprotective effect...
The neurotoxicity of aggregated amyloid ${\beta}$ ($A{\beta}$) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD). It can cause neurotoxicity in AD by evoking a cascade of apoptosis to neuron. Here, we investigated the neuroprotective effects of cilostazol, which acts as a phosphodiesterase III inhibitor, on $A{\beta}_{25-35}$-induced cytotoxicity in mouse neuronal cells and cognitive decline in the C57BL/6J AD mouse model via peroxisome proliferator-activated receptor (PPAR)-${\gamma}$ activation. $A{\beta}_{25-35}$ significantly reduced cell viability and increased the number of apoptotic-like cells. Cilostazol treatment recovered cells from $A{\beta}$-induced cell death as well as rosiglitazone, a PPAR-${\gamma}$ activator. These effects were suppressed by GW9662, an antagonist of PPAR-${\gamma}$ activity, indicative of a PPAR-${\gamma}$-mediated signaling. In addition, cilostazol and rosiglitazone also restored PPAR-${\gamma}$ activity levels that had been altered as a result of $A{\beta}_{25-35}$ treatment, which were antagonized by GW9662. Furthermore, cilostazol also markedly decreased the number of apoptotic-like cells and decreased the Bax/Bcl-2 ratio. Intracerebroventricular injection of $A{\beta}_{25-35}$ in C57BL/6J mice resulted in impaired cognitive function. Oral administration of cilostazol (20 mg/kg) for 2 weeks before $A{\beta}_{25-35}$ injection and once a day for 4 weeks post-surgery almost completely prevented the $A{\beta}_{25-35}$-induced cognitive deficits, as did rosiglitazone. Taken together, our findings suggest that cilostazol could attenuate $A{\beta}_{25-35}$-induced neuronal cell injury and apoptosis as well as promote the survival of neuronal cells, subsequently improving cognitive decline in AD, partly because of PPAR-${\gamma}$ activation. The phosphodiesterase III inhibitor cilostazol may be the basis of a novel strategy for the therapy of AD.
The neurotoxicity of aggregated amyloid ${\beta}$ ($A{\beta}$) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD). It can cause neurotoxicity in AD by evoking a cascade of apoptosis to neuron. Here, we investigated the neuroprotective effects of cilostazol, which acts as a phosphodiesterase III inhibitor, on $A{\beta}_{25-35}$-induced cytotoxicity in mouse neuronal cells and cognitive decline in the C57BL/6J AD mouse model via peroxisome proliferator-activated receptor (PPAR)-${\gamma}$ activation. $A{\beta}_{25-35}$ significantly reduced cell viability and increased the number of apoptotic-like cells. Cilostazol treatment recovered cells from $A{\beta}$-induced cell death as well as rosiglitazone, a PPAR-${\gamma}$ activator. These effects were suppressed by GW9662, an antagonist of PPAR-${\gamma}$ activity, indicative of a PPAR-${\gamma}$-mediated signaling. In addition, cilostazol and rosiglitazone also restored PPAR-${\gamma}$ activity levels that had been altered as a result of $A{\beta}_{25-35}$ treatment, which were antagonized by GW9662. Furthermore, cilostazol also markedly decreased the number of apoptotic-like cells and decreased the Bax/Bcl-2 ratio. Intracerebroventricular injection of $A{\beta}_{25-35}$ in C57BL/6J mice resulted in impaired cognitive function. Oral administration of cilostazol (20 mg/kg) for 2 weeks before $A{\beta}_{25-35}$ injection and once a day for 4 weeks post-surgery almost completely prevented the $A{\beta}_{25-35}$-induced cognitive deficits, as did rosiglitazone. Taken together, our findings suggest that cilostazol could attenuate $A{\beta}_{25-35}$-induced neuronal cell injury and apoptosis as well as promote the survival of neuronal cells, subsequently improving cognitive decline in AD, partly because of PPAR-${\gamma}$ activation. The phosphodiesterase III inhibitor cilostazol may be the basis of a novel strategy for the therapy of AD.
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가설 설정
To explore the molecular mechanisms underlying Aβ 25-35-induced cell apoptosis, we examined the expression of Bcl-2 and Bax to determine whether the regulation of these cell death-associated proteins might be responsible for the protective effect of cilostazol.
We hypothesized that the phosphodiesterase III inhibitor cilostazol decreased neuronal cell death via PPAR-γ activation, subsequently improving the cognitive decline in AD.
제안 방법
Aβ25-35 significantly increased escape latency time at 4 weeks after Aβ25-35 injection (p<0.05 vs control, Two-way ANOVA), which were reversed by cilostazol (p<0.05 vs vehicle, Two-way ANOVA).
For each trial, mice were randomized to one of four directional starting locations (north, south, east and west) and were placed in the pool facing the wall.
Spatial learning and memory deficits were assessed using the Morris water maze task as described previously [3,30] with minor modification. The experiment was performed on mice after 4 weeks Aβ25-35 injection period.
We investigated the preventive effect of cilostazol, by administering cilostazol beginning 2 weeks before Aβ25-35 injection and once a day for 4 weeks post-surgery.
대상 데이터
The experiment was performed on mice after 4 weeks Aβ25-35 injection period. The maze consisted of a 1.15 m-diameter pool which was painted flat white. A 10 cm-diameter platform was placed halfway between the center of the pool and the edge, and was positioned 1 cm below the surface of the water.
데이터처리
Statistical comparisons were performed using paired or unpaired Student’s t test and one-way analysis of variance (ANOVA) or two-way ANOVA for repeated measures followed by Fisher’s protected least significant difference test.
이론/모형
After this incubation, PPARγ transcription activity was determined using Luciferase reporter assay.
(A) Mouse neuronal cells treated with or without cilostazol (Cilo; 1 30 μM), followed by incubation with 30 μM Aβ25-35 for 24 hr. After this incubation, cell viability was determined using the MTT assay. (B) The effect of 30 μM cilostazol against Aβ-induced neurotoxicity was assessed in the absence and presence of 5 μM GW9662 (GW) compared with 10 μM rosiglitazone (Rosi).
성능/효과
In conclusion, a phosphodiesterase III inhibitor cilostazol rescues neuronal cells from Aβ25-35-induced cell death and improves cognitive decline in AD mouse model through activation of PPAR-γ transcription. Further study of the anti-apoptotic and anti-inflammatory properties of cilostazol may provide opportunities for novel pharmacological interventions aimed at preventing or palliating the consequences of AD.
The ratio of Bax to Bcl-2 has been reported to be correlated with apoptosis. Our results showed that cilostazol markedly decreased Bax/Bcl-2 ratio in a similar degree as did rosiglitazone, both of which were significantly inhibited by GW9662. The results suggest that cilostazol could prevent Aβ25-35-induced apoptosis, at least in part, via PPAR-γ-mediated regulation of Bcl-2 and Bax expression.
The results suggested that cilostazol could prevent Aβ25-35-induced apoptosis, at least in part, via PPAR-γ-mediated regulation of Bcl-2 and Bax expression.
Thus, the results of the MTT assay showed that cilostazol could block cytotoxic effects of Aβ25-35 via PPAR-γ activation in mouse neuronal cells.
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