The apoptotic effects of oxidative stress and antiapoptotic effects of caspase inhibitors on rat notochordal cells
The apoptotic effects of oxidative stress and antiapoptotic effects of caspase inhibitors on rat notochordal cells
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DOI：
10.1097/brs.0b013e318157395a
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发表时间：
2007-10-15
期刊：
影响因子：
3
通讯作者：
Woo, Young-Kyun
中科院分区：
文献类型：
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作者：
Kim, Ki-Won;Ha, Kee-Yong;Woo, Young-Kyun
研究设计。利用大鼠脊索细胞进行Western blotting和流式细胞仪分析。目的：探讨氧化应激对大鼠脊索细胞的促凋亡作用及半胱氨酸氨基转移酶抑制剂的抗凋亡作用。尽管氧化应激可引起多种细胞类型的凋亡，但其对脊索细胞凋亡的影响以及caspase抑制剂对氧化应激诱导的细胞凋亡的拮抗作用尚不清楚。培养的大鼠脊索细胞暴露于氧化应激[500mU/L的过氧化氢(H_2O_2)]。用免疫印迹法检测氧化应激6h后半胱氨酸天冬氨酸氨基转移酶(-3、-8、-9)的激活以及Bid和多聚ADP-核糖聚合酶(PARP)的裂解，以确定氧化应激诱导的细胞凋亡途径。为了阐明caspase抑制剂在氧化应激诱导的细胞凋亡中的抗凋亡作用，用流式细胞术检测了caspase-8的z-IETD-fmk、caspase-9的z-Lehd-fmk和caspase-3的z-DEVD-fmk处理前后脊索细胞的凋亡率。氧化应激显著增加大鼠脊索细胞凋亡率(2.1%vs.4.75%，P=0.008)，并导致内源性(caspase-9)和外源性(caspase-8)通路的启动子及其共同执行者(caspase-3)的激活。它还导致了BID和PARP的裂解。流式细胞仪分析显示，caspase-9抑制剂(4.75%vs.3.56%，P=0.31)和caspase-8抑制剂(4.75%vs.5.24%，P=0.84)仅抑制内源和外源途径中的一个，但不能显著抑制氧化应激诱导的细胞凋亡。而半胱氨酸天冬氨酸氨基转移酶3抑制剂抑制这两条通路后，氧化应激诱导的细胞凋亡率显著减少(4.75%vs.2.64%，P=0.008)至对照组水平(2.1%vs.2.64%，P=0.15)。氧化应激通过内源性和外源性(I型和II型)途径引起大鼠脊索细胞的凋亡。由于caspase抑制剂正被用于临床试验，使用caspase抑制剂抑制这两条通路在氧化应激诱导脊索细胞凋亡中可能具有未来的治疗重要性。我们的结果表明，抑制不适当或过早的氧化应激诱导的脊索细胞凋亡可能会推迟椎间盘退变的起点。
Study Design. Western blotting and flow cytometric analyses were performed using rat notochordal cells.Objective. To demonstrate the apoptotic effect of oxidative stress and the antiapoptotic effects of caspase inhibitors on rat notochordal cells.Summary of Background Data. Although oxidative stress causes apoptosis in many cell types, its effect on the apoptosis of notochordal cell and antiapoptotic effects of caspase inhibitors on the oxidative stress-induced apoptosis are unknown.Methods. Cultured rat notochordal cells were exposed to oxidative stress [500 mu mol/L of hydrogen peroxide (H2O2)]. To determine the oxidative stress-induced apoptotic pathways, activations of caspases (-3, -8, and -9) as well as cleavages of Bid and poly (ADP-ribose) polymerase ( PARP) were evaluated with Western blotting 6 hours after oxidative stress. To elucidate the antiapoptotic effects of caspase inhibitors on the oxidative stress induced-apoptosis, apoptotic rates of notochordal cells with or without treatment of specific caspase inhibitors (z-IETD-fmk for caspase-8, z-LEHD-fmk for caspase-9, and z-DEVD-fmk for caspase-3) were quantified by flow cytometry.Results. Oxidative stress significantly increased apoptosis of rat notochordal cells (2.1% vs. 4.75%, P = 0.008) and led to activations of initiators of intrinsic (caspases-9) and extrinsic (caspase-8) pathways as well as their common executioner (caspase-3). It also caused cleavages of Bid and PARP. Flow cytometric analysis showed that inhibition of only one of the intrinsic and extrinsic pathways by caspase-9 inhibitor (4.75% vs. 3.56%, P = 0.31) and caspase-8 inhibitor (4.75% vs. 5.24%, P = 0.84) did not significantly suppress the oxidative stress-induced apoptosis. However, inhibition of both pathways by caspase-3 inhibitor significantly reduced the oxidative stress-induced apoptosis (4.75% vs. 2.64%, P = 0.008) to the control level (2.1% vs. 2.64%, P = 0.15).Conclusion. Oxidative stress caused apoptosis of rat notochordal cells via both intrinsic and extrinsic (Type I and Type II) pathways. Because caspase inhibitors are being used in clinical trials, inhibition of both pathways using caspase inhibitors might be of future therapeutic importance in oxidative stress-induced apoptosis of notochordal cells. Our results suggest that inhibition of inappropriate or premature oxidative stress-induced apoptosis of notochordal cells may delay the starting point of disc degeneration.