Propofol and magnesium attenuate isoflurane-induced caspase-3 activation via inhibiting mitochondrial permeability transition pore

We employed H4 human neuroglioma cells, stably transfected to express full-length (FL) amyloid precursor protein (APP) (H4-APP cells) in the experiments. The cells were cultured in Dulbecco's Modified Eagle Media (DMEM) containing 9% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine, and were supplemented with 220 μg/ml G418.

Cells were treated with 2% isoflurane plus 21% O₂ and 5% CO₂ for six hours as described by our previous studies [41] for the purpose of measuring caspase-3 activation. The cultured cells were treated for three hours in the studies to measure mPTP opening as described by our precious studies [24]. Treatment with 2% isoflurane for three hours may not induce caspase-3 activation and apoptosis (Figure 1). Thus, we assessed whether the treatment with 2% isoflurane for three hours might induce opening of mPTP without causing caspase-3 activation in the cells. In the interaction experiments, 50 μM magnesium sulfate (Mg²⁺) or 200 μM propofol was administrated to the cells one hour before the isoflurane treatment as well as during isoflurane treatment.

C57BL/6 J mice (The Jackson Laboratory, Bar Harbor, ME) were used in the experiments as described before [17, 42]. The animal protocol was approved by Standing Committee on Animals at Massachusetts General Hospital (Boston, MA). The mice were randomized by weight and gender into experimental groups that received 1.4% isoflurane plus 100% oxygen for six hours, and control groups that received 100% oxygen for six hours at identical flow rates in identical anesthetizing chambers. Anesthetic and oxygen concentrations were measured continuously (Datex, Tewksbury, MA), and the temperature of the anesthetizing chamber was controlled to maintain the rectal temperature of the mice at 37 ± 0.5°C. In the interaction studies, Mg²⁺ (100 mg/kg) or propofol (50 mg/kg) was administered to the mice via intraperitoneal injection 10 minutes before the isoflurane anesthesia. 200 μM propofol has been shown to have neuroprotective effects in an in vitro model of traumatic brain injury[43]; we therefore used this concentration of propofol to determine whether propofol can attenuate the isoflurane-induced mPTP opening. 50 and 100, but not 25, mg/kg propofol have been shown to produce neuroprotection effects in ischemic mice models [44]. Thus, we used 50 mg/kg propofol in the current studies. And we used 100 mg/kg Mg²⁺ on mice because Mg²⁺ has been shown to have a neuroprotective effect on cerebral ischemia [45]. And based on our preliminary results, we used 50 μM Mg²⁺ in the in vitro the studies. Whole brain tissues of mice were harvested at end of the anesthesia.

The harvested brain tissues were homogenized on ice using an immunoprecipitation buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40) plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A). The lysates were collected, centrifuged at 13,000 rpm for 15 min, and quantified for total proteins by a bicinchoninic acid protein assay kit (Pierce, Iselin, NJ).

The harvested H4-APP cells and brain tissues were subjected to Western blot analyses as described by Xie et al. [17, 18, 20] and Zhang et al. [46, 47]. A caspase-3 antibody (1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA) was used to recognize FL-caspase-3 (35 – 40 kDa) and caspase-3 fragment (17 – 20 kDa) resulting from cleavage at asparate position 175. Antibody anti-β-Actin (1:10,000, Sigma, St. Louis, MO) was used to detect β-Actin (42 kDa). Each band in the Western blot represented an independent experiment. The results were averaged from three to 8 independent experiments. Briefly, the intensity of the signals was analyzed using the National Institute of Health image program (National Institute of Health Image 1.62, Bethesda, MD). The caspase-3 normalization was performed by determining the ratio of caspase-3 fragment to FL caspase-3. Then, the changes in levels of caspase-3 in treated cells or mice were presented as percentages of the corresponding levels in control cells or mice.

H4-APP cells were treated with 2% isoflurane for three hours. For the interaction studies, 50 μM Mg²⁺ or 200 μM propofol was administrated to cells one hour before the isoflurane treatment. The opening of mPTP was determined by flowcytometry, using the MitoProbe^TM Transition Pore Assay Kit (Invitrogen, Carlsbad, CA). In normal conditions, the non-fluorescent acetoxymethyl ester (AM) of calcein dye (calcein AM) and cobalt can enter the cell. The acetoxymethyl ester (AM) groups are cleaved from calcein via non-specific esterase, and calcein can then show fluorescence signals in both the cytosol and mitochondria. Cobalt can quench the cytosolic calcein signal. However, cobalt cannot enter healthy mitochondria freely, and therefore cannot quench the mitochondrial calcein signal. When opening of mPTP occurs, cobalt enters through the pore and subsequently quenches the mitochondrial calcein signal. Flowcytometry was used to detect the amount of cells that exhibit quenched calcein signals inside the mitochondria. The location of the curves indicates the amount of such cells, which suggests the opening of mPTP. Ionomycin was used as a positive control for the opening of mPTP in the experiments. Dead cells and debris were excluded from analysis by gates set on forward and side angle light scatter.

Given the presence of background caspase 3 activation in cells and brain tissues of mice, we did not use absolute values to describe these changes. Instead, these changes were presented as percentages of those from the control group. For example, one hundred percent of caspase-3 activation refers to the control level for the purpose of comparison to experimental conditions. Data were expressed as mean ± S.D. The number of samples varied from three to 8, and the samples were normally distributed. We used a two-tailed t-test to compare the difference between the control condition and isoflurane treatment, and the difference between propofol, Mg²⁺ and their controls. P-values less than 0.05 (* or #) and 0.01 (** or ##) were considered statistically significant.

The H4-APP cells were treated with 50 μM Mg²⁺ or saline for 10 minutes followed by 2% isoflurane or control condition for six hours. The cells were harvested at the end of the experiment and were subjected to Western blot analysis. Caspase-3 immunoblotting revealed that the isoflurane treatment induced caspase-3 activation (Figure 2A) as evidenced by increased ratios of cleaved (activated) caspase-3 fragment (17 kDa) to full-length (FL) (35–40 kDa) caspase-3. Treatment with 50 μM Mg²⁺ alone did not induce caspase-3 activation, but the Mg²⁺ treatment attenuated the isoflurane-induced caspase-3 activation (Figure 2A). Quantification of the Western blots (Figure 2B), based on the ratio of caspase-3 fragment to FL caspase-3, revealed that isoflurane (black bar) led to caspase-3 activation as compared to the control condition (white bar): 1.54 versus 1.00 fold (** P = 0.001). The Mg²⁺ treatment (net bar) attenuated the isoflurane-induced caspase-3 activation: 1.23 fold versus 1.54 fold (# P = 0.03). These findings suggest that Mg²⁺ may mitigate the isoflurane-induced caspase-3 activation in H4-APP cells.

Next, we performed the in vivo relevance studies by assessing the effects of isoflurane and Mg²⁺ on caspase-3 activation in the brain tissues of six-day old WT mice. As can be seen in Figure 2C, Mg²⁺ (lane 4) attenuated the isoflurane-induced caspase-3 activation (lane 3) in the brain tissues of the mice. The Mg²⁺ treatment alone (lane 2) did not induce caspase-3 activation as compared to the saline group (lane 1) in the brain tissues of the mice. Quantification of the Western blot further illustrated that the isoflurane (black bar) led to caspase-3 activation as compared to the control condition (white bar): 1.52 versus 1.00 fold (* P = 0.02). Mg²⁺ treatment (net bar) attenuated the isoflurane-induced caspase-3 activation (black bar): 1.38 versus 1.52 fold (# P = 0.03), (Figure 2D). These results from the in vivo studies further suggest that Mg²⁺ may attenuate the isoflurane-induced caspase-3 activation.

Our previous studies have illustrated that propofol can attenuate the isoflurane-induced caspase-3 activation in H4-APP cells. [48]. In the current experiments, we performed the in vivo relevance studies by assessing the effects of isoflurane and propofol on caspase-3 activation in the brain tissues of WT six-day old mice. As can be seen in Figure 3A, propofol (lane 10–11) attenuated the isoflurane-induced caspase-3 activation (lane 7–9) in the brain tissues of the mice. The propofol treatment alone (lane 4–6) did not induce caspase-3 activation compared with the saline group (lane 1–3) in the brain tissues of the mice. Quantification of the Western blot further illustrated that the isoflurane anesthesia (black bar) led to caspase-3 activation as compared to the control condition (white bar): 1.33 versus 1.00 fold (** P = 0.01). Propofol treatment (net bar) attenuated the isoflurane-induced caspase-3 activation in the mice (black bar): 1.20 versus 1.33 fold (# P = 0.02), (Figure 3B). These results from the in vivo studies further suggest that propofol may attenuate the isoflurane-induced caspase-3 activation.

Given that Mg²⁺ and propofol can attenuate the isoflurane-induced caspase-3 activation, and the isoflurane-induced caspase-3 activation may result from the isoflurane-induced opening of mPTP, next, we asked whether Mg²⁺ and propofol, the blockers of mPTP opening, can attenuate the isoflurane-induced mPTP opening.

Flow cytometric analysis of calceinAM and cobalt showed that the treatment with 50 μM Mg²⁺ (Figure 4, peak 3) led to reductions in the isoflurane-induced mPTP opening (Figure 4, peak 2), as evidenced by the right-shift of the curve, whereas the Mg²⁺ treatment alone did not affect the opening of mPTP in H4-APP cells (data not shown). Next, we found that the treatment with 50 μM propofol (Figure 5, peak 3) led to reductions in the isoflurane-induced mPTP opening (Figure 5, peak 2), whereas the propofol treatment alone did not affect the opening of mPTP in H4-APP cells (data not shown). Taken together, these findings suggested that Mg²⁺ and propofol may mitigate the isoflurane-induced caspase-3 activation by inhibiting the isoflurane-induced opening of mPTP.