The mitochondrial pathway of anesthetic isoflurane-induced apoptosis

Abstract

The common inhalation anesthetic isoflurane has been shown to induce apoptosis, which then leads to accumulation of beta-amyloid protein, the hallmark feature of Alzheimer disease neuropathogenesis. The underlying molecular mechanism of the isoflurane-induced apoptosis is largely unknown. We, therefore, set out to assess whether isoflurane can induce apoptosis by regulating Bcl-2 family proteins, enhancing reactive oxygen species (ROS) accumulation, and activating the mitochondrial pathway of apoptosis. We performed these studies in cultured cells, primary neurons, and mice. Here we show for the first time that treatment with 2% isoflurane for 6 h can increase pro-apoptotic factor Bax levels, decrease anti-apoptotic factor Bcl-2 levels, increase ROS accumulation, facilitate cytochrome c release from the mitochondria to the cytosol, induce activation of caspase-9 and caspase-3, and finally cause apoptosis as compared with the control condition. We have further found that isoflurane can increase the mRNA levels of Bax and reduce the mRNA levels of Bcl-2. The isoflurane-induced ROS accumulation can be attenuated by the intracellular calcium chelator BAPTA. Finally, the anesthetic desflurane does not induce activation of mitochondrial pathway of apoptosis. These results suggest that isoflurane may induce apoptosis through Bcl-2 family proteins- and ROS-associated mitochondrial pathway of apoptosis. These findings, which have identified at least partially the molecular mechanism by which isoflurane induces apoptosis, will promote more studies aimed at studying the potential neurotoxic effects of anesthetics.

FIGURE 1.

Isoflurane increases Bax levels in H4-APP cell and primary neurons from naïve mice. A, treatment with 2% isoflurane for 6 h (lanes 7–12) increased Bax levels as compared with the control condition (lanes 1–6) in the H4-APP cells. There was no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated cells. B, quantification of the Western blot shows that isoflurane treatment (black bar) increases Bax levels compared with the control condition (white bar), normalized to β-actin levels (*, p = 0.04). C, treatment with 2% isoflurane for 6 h (lanes 3–6) increased Bax levels as compared with the control condition (lanes 1 and 2) in the primary neurons from naïve mice. There was no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated neurons. D, quantification of the Western blot shows that isoflurane treatment (black bar) increased Bax levels compared with the control condition (white bar), normalized to β-actin levels (**, p = 0.008). E, treatment with 2% isoflurane for 6 h (black bar) increased the mRNA levels of Bax as compared with the control condition (white bar) in the primary neurons from naïve mice (*, p = 0.02).

FIGURE 2.

Isoflurane decreases Bcl-2 levels in H4-APP cells, primary neurons from naïve mice, and brain tissues of naïve mice. A, treatment with 2% isoflurane for 6 h (lanes 5–8) decreased Bcl-2 levels as compared with the control conditions (lanes 1–4) in the H4-APP cells. There was no significant difference in the amounts of β-actin under the control condition- or isoflurane-treated cells. B, quantification of the Western blot shows that isoflurane treatment (black bar) decreased Bcl-2 levels compared with the control conditions (white bar), normalized to β-actin levels (*, p = 0.017). C, treatment with 2% isoflurane for 6 h (lanes 3 and 4) decreased Bcl-2 levels as compared with the control conditions (lanes 1 and 2) in the primary neurons from naïve mice. There was no significant difference in amounts of β-actin in the control condition- or isoflurane-treated neurons. D, quantification of the Western blot shows that the isoflurane treatment (black bar) decreased Bcl-2 levels compared with the control condition (white bar), normalized to β-actin levels (*, p = 0.027). E, treatment with 2% isoflurane for 6 h (black bar) decreased the mRNA levels of Bcl-2 as compared with the control condition (white bar) in the primary neurons from naïve mice (*, p = 0.049). F, treatment with 1.4% isoflurane for 2 h (lanes 4–6) decreased Bcl-2 levels as compared with the control conditions (lanes 1–3) in brain tissues of naïve mice. There was no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated naïve mice. G, quantification of the Western blot shows that isoflurane treatment (black bar) decreased Bcl-2 levels compared with the control condition (white bar), normalized to β-actin levels (**, p = 0.004).

FIGURE 3.

Isoflurane induces ROS accumulation in the mitochondria of H4-APP Cells. A, column 1 is the image of mitochondria (red), column 2 is the image of ROS (green) both inside and outside of the mitochondria, and column 3 is the merged image. The orange color, but not red color, in the merged images (column 3) indicates that ROS is inside the mitochondria. Row a shows cells following the control condition, and the row b shows cells treated with 2% isoflurane for 6 h. The treatment with isoflurane (row b) led to accumulation of ROS inside the mitochondria (orange color) as compared with the control condition (row a, red color). B, a fluorescence assay shows that isoflurane treatment (black bar) increases ROS accumulation compared with the control condition (white bar) (**, p = 0.006). C, treatment with BAPTA plus isoflurane (net bar) induced a lesser degree of ROS accumulation compared with a treatment with DMSO plus isoflurane (black bar) (*, p = 0.025).

FIGURE 4.

Isoflurane facilitates the release of cytochrome c from the mitochondria to the cytosol in H4-APP cells. A, column 1 is the image of mitochondria (red), column 2 is the image of cytochrome c (green) both inside and outside mitochondria, column 3 is the image of nuclei (blue), and column 4 is the merged image. The orange color in the merged images (column 4) indicates the existence of cytochrome c inside the mitochondria, and the green dots in the merged images indicate the existence of cytochrome c outside the mitochondria. Row a shows cells following the control condition, row b show cells treated with STS, the positive control of the experiment, and the row c show cells treated with 2% isoflurane for 6 h. The treatment with STS (row b) or isoflurane (row c) leads to the appearance of cytochrome c outside of the mitochondria (green dots, indicated by the arrows) as compared with the control condition (row a). B, treatment with 2% isoflurane for 6 h (lanes 5–8) increased cytochrome c levels in cytosol as compared with the control condition (lanes 1–4) in H4-APP cells. There was no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated H4-APP cells. C, quantification of the Western blot shows that isoflurane treatment (black bar) increased cytochrome c levels in cytosol compared with the control condition (white bar), normalized to β-actin levels (*, p = 0.01).

FIGURE 5.

Isoflurane induces activation of caspase-9 and caspase-3, but not caspase-8, in H4-APP cells and primary neurons from naïve mice. A, treatment with 2% isoflurane for 6 h (lanes 4–6) induced caspase-9 cleavage (activation) as compared with the control condition (lanes 1–3) in H4-APP cells. There was significant difference in the amounts of β-actin in the control condition- or the isoflurane-treated H4-APP cells. B, treatment with 2% isoflurane for 6 h (lanes 3–5) did not induce caspase-8 cleavage (activation) as compared with the control conditions (lanes 1 and 2) in H4-APP cells. Serum deprivation treatment (lane 6), the positive control in the experiment, increased the levels of the caspase-8 fragment (both 18 and 42 kDa). There was no significant difference in amounts of β-actin in the control condition- or isoflurane-treated H4-APP cells. C, treatment with 2% isoflurane for 6 h (lanes 4–6) induced caspase-3 cleavage (activation) as compared with the control condition (lanes 1–3) in the primary neurons from naïve mice. There was no significant difference in amounts of β-actin in the control condition- or in the isoflurane-treated neurons. FL, full-length.

FIGURE 6.

Isoflurane increases TUNEL-positive H4-APP cells. A, treatment with 2% isoflurane for 6 h increased TUNEL-positive cells as compared with the control condition in H4-APP cells. B, quantification of the TUNEL-positive cells illustrates that the isoflurane treatment (black bar) increases the amounts of TUNEL-positive H4-APP cells as compared with the control condition (white bar) (**, p = 0.002).

FIGURE 7.

Desflurane does not alter the levels of Bax, Bcl-2, ROS, cytochrome c, and caspase-9 in H4-APP cells. Treatment with 12% desflurane for 6 h did not affect the levels of Bax (A), Bcl-2 (B), ROS (C), cytochrome c (D), and caspase-9 (E).

FIGURE 8.

Hypothetical pathway by which isoflurane induces apoptosis. Isoflurane increases pro-apoptotic factor Bax levels, decreases anti-apoptotic factor Bcl-2 levels, and enhances ROS accumulation to induce mitochondrial damage. The damaged mitochondria release cytochrome c, which activates caspase-9. The activated caspase-9 then induces caspase-3 activation, leading to apoptosis.