Kim Ki-Won, Ha Kee-Yong, Lee Jun-Seok, Rhyu Kee-Won, An Howard S, Woo Young-Kyun
Department of Orthopedic Surgery, St. Mary's Hospital, College of Medicine, Catholic University of Korea, Seoul, Korea.
Spine (Phila Pa 1976). 2007 Oct 15;32(22):2443-8. doi: 10.1097/BRS.0b013e318157395a.
Western blotting and flow cytometric analyses were performed using rat notochordal cells.
To demonstrate the apoptotic effect of oxidative stress and the antiapoptotic effects of caspase inhibitors on rat notochordal cells.
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.
Cultured rat notochordal cells were exposed to oxidative stress [500 micromol/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.
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).
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.
使用大鼠脊索细胞进行蛋白质印迹法和流式细胞术分析。
证明氧化应激对大鼠脊索细胞的凋亡作用以及半胱天冬酶抑制剂的抗凋亡作用。
尽管氧化应激在许多细胞类型中会导致细胞凋亡，但其对脊索细胞凋亡的影响以及半胱天冬酶抑制剂对氧化应激诱导的细胞凋亡的抗凋亡作用尚不清楚。
将培养的大鼠脊索细胞暴露于氧化应激（500微摩尔/升过氧化氢）。为了确定氧化应激诱导的凋亡途径，在氧化应激6小时后，通过蛋白质印迹法评估半胱天冬酶（-3、-8和-9）的激活以及Bid和聚（ADP-核糖）聚合酶（PARP）的裂解。为了阐明半胱天冬酶抑制剂对氧化应激诱导的细胞凋亡的抗凋亡作用，通过流式细胞术对未处理或经特定半胱天冬酶抑制剂（针对半胱天冬酶-8的z-IETD-fmk、针对半胱天冬酶-9的z-LEHD-fmk和针对半胱天冬酶-3的z-DEVD-fmk）处理的脊索细胞的凋亡率进行定量。
氧化应激显著增加大鼠脊索细胞的凋亡（2.1%对4.75%，P = 0.008），并导致内源性（半胱天冬酶-9）和外源性（半胱天冬酶-8）途径的起始因子及其共同的执行者（半胱天冬酶-3）激活。它还导致Bid和PARP的裂解。流式细胞术分析表明，半胱天冬酶-9抑制剂（4.75%对3.56%，P = 0.31）和半胱天冬酶-8抑制剂（4.75%对5.24%，P = 0.84）仅抑制内源性或外源性途径之一，并未显著抑制氧化应激诱导的细胞凋亡。然而，半胱天冬酶-3抑制剂同时抑制两条途径可显著降低氧化应激诱导的细胞凋亡（4.75%对2.
