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Spine (Phila Pa 1976). 2007 Oct 15;32(22):2443-8.

The apoptotic effects of oxidative stress and antiapoptotic effects of caspase inhibitors on rat notochordal cells.

Author information

1
Department of Orthopedic Surgery, St. Mary's Hospital, College of Medicine, Catholic University of Korea, Seoul, Korea.

Abstract

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 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.

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.

PMID:
18090083
DOI:
10.1097/BRS.0b013e318157395a
[Indexed for MEDLINE]

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