虚血性脳卒中ラットの神経細胞アポトーシスとErk1/2シグナル経路の関係

Relationship between Erk1/2 signal pathway and nerve cell apoptosis rats with ischemic stroke.

• Xiaorong Gao
• Haijun Li
• Zhiyan Dong
• Pengxuan Zhang
• Rui Liu
• Yidong Xue
• Ning Shi

抄録

虚血性脳卒中ラットにおけるErk1/2シグナル経路と神経細胞のアポトーシスとの関係を調べる。雄性SD(Sprague Dawley)ラット(n = 24)を無作為に3群に分け、それぞれ8匹ずつ、偽手術群、MCAO(Midle cerebral artery oclusion)群、MCAO+U0126介入群(U0126群)とした。in vitro試験では、初代皮質神経細胞をコントロール群、OGD(Oxygen and glucose deprivation)群、U0126介入群(U0126群)の3群に分けて試験を行った。Erk1/2、p-Erk1/2、Bcl-2のin vivoでのタンパク質発現量をウエスタンブロットを用いて測定した。Bcl-2、Bcl-xlおよびBaxの発現は、免疫組織化学染色を用いてアッセイした。脳組織における神経細胞死は、TUNEL染色を用いて検出した。インビトロ試験では、細胞のアポトーシスをフローサイトメトリーおよびLDH放出を用いてアッセイした。カスパーゼ-３の活性を決定した。神経細胞のアポトーシスは、Hoechst33258染色法を用いて決定した。in vivo試験では、MCAO群の脳組織におけるp-ERK1/2のタンパク質発現量は、偽手術群と比較して有意に増加したが、U0126群のp-ERK1/2のタンパク質発現量はMCAO群と比較して有意に低いことがわかった。MCAO群のBcl-2およびBcl-xlの発現レベルは、偽手術群の対応する発現レベルと比較して有意に低かったが、U0126群のBcl-2およびBcl-xlの発現レベルは、MCAO群の発現レベルと比較して有意に低かった。MCAO群では、偽手術群よりもBaxの発現量が有意に高かったのに対し、U0126群ではMCAO群よりもBaxの発現量が高かった。神経細胞の死滅数は、MCAO群では偽手術群に比べて有意に多く、U0126群ではMCAO群に比べて神経細胞の死滅数が有意に少なかった。in vitro試験では、フローサイトメトリーの結果、対照群と比較してOGD処理した神経細胞のアポトーシスが有意に高かった。U0126に曝露し、ODR（酸素依存性リプレッサー）で処理した神経細胞は、OGD単独投与群と比較して有意に個体数が減少した。OGD処理した神経細胞のLDH放出レベルは、対照群と比較して有意に増加した。しかし、U0126介入後にOGD処理した神経細胞のLDH放出レベルは、単一のOGD処理群と比較して有意に減少した。OGD処置後の神経細胞核の希釈度は、対照群と比較して有意に増加した。Ｕ０１２６介入後にＯＤＲで処理した神経細胞では、ＯＧＤ単独投与群の神経細胞核の希釈度と比較して、有意に減少した。OGD治療は、対照群と比較して、神経細胞のカスパーゼ-３活性を有意に増加させた。しかし、U0126介入後にODRを投与した神経細胞のカスパーゼ-3活性は、OGD単独投与群と比較して有意に低下した。虚血性脳卒中におけるErk1/2シグナル経路の活性化は、神経細胞のアポトーシスを促進することが示唆された。これらの知見から、ERK1/2シグナル経路は虚血性脳卒中の治療において重要な標的である可能性が合理的に推察される。

To investigate the relationship between the Erk1/2 signal pathway and neuronal apoptosis in ischemic stroke rats. Male SD(Sprague Dawley)  rats (n = 24) were randomly divided into three groups, each containing 8 rats: sham-operated group, MCAO(Midle cerebral artery oclusion)  group, and MCAO + U0126 intervention group (U0126 group). In in vitro trial, primary cortical nerve cells were divided into three groups: control group, OGD(Oxygen and glucose deprivation)  group, and U0126 intervention group (U0126 group). In vivo protein expression levels of Erk1/2, p-Erk1/2 and Bcl-2 were determined using western blot. The expressions of Bcl-2, Bcl-xl and Bax were assayed using immunohistochemical staining. Nerve cell mortality in cerebral tissue was detected using TUNEL staining. In in vitro trials, cell apoptosis was assayed with flow cytometry and LDH release. The activity of caspase-3 was determined. Nerve cell apoptosis was determined using Hoechst33258 staining method. In in vivo trial, it was found that the protein expression level of p-ERK1/2 in cerebral tissue in the MCAO group was significantly increased, when compared with that of the sham-operated group, while the protein expression level of p-Erk1/2 in the U0126 group was significantly lower than that in the MCAO group. The expression levels of Bcl-2 and Bcl-xl in the MCAO group were significantly lower than the corresponding expression levels in the sham-operated group, while the expressions of Bcl-2 and Bcl-xl in the U0126 group were significantly lower than those in MCAO group. In MCAO group, the expression of Bax was significantly higher than that in the sham-operated group, while Bax expression was higher in U0126 than in MCAO group. There were significantly higher number of dead nerve cells in MCAO group than in the sham-operated group, while nerve cell mortality in U0126 group was significantly lower than in MCAO group. In in vitro trials, flow cytometry revealed significantly higher apoptosis of OGD-treated nerve cells, relative to the control group. Nerve cells exposed to U0126 and treated with ODR (Oxygen-dependent repressor)  were significantly decreased in population, when compared with single OGD treatment group. The LDH release level of nerve cells treated OGD was significantly increased, when compared with that of the control group. However, LDH release level of nerve cells treated with OGD after U0126 intervention was significantly decreased, relative to the single OGD treatment group. The dilution of nerve cell nucleus after OGD treatment was significantly increased, when compared with that of the control group. For nerve cells treated with ODR after U0126 intervention, the nuclear dilution was significantly decreased, relative to that of nerve cell nucleus in the single OGD treatment group. The OGD treatment led to significant increase in nerve cell caspase-3 activity, relative the control group. However, the caspase-3 activity of nerve cells treated with ODR after U0126 intervention was significantly decreased, when compared with single OGD treatment group. The activation of Erk1/2 signal pathway during ischemic stroke promotes apoptosis of nerve cells. Based on these findings, it can be reasonably inferred that the ERK1/2 signal pathway may be an important target for treating ischemic stroke.