Acetaminophen protects brain endothelial cells against oxidative stress

Abstract

Increasing evidence suggests that acetaminophen has unappreciated anti-oxidant and anti-inflammatory properties. Drugs that affect oxidant and inflammatory stress in the brain are of interest because both processes are thought to contribute to the pathogenesis of neurodegenerative disease. The objective of this study is to determine whether acetaminophen affects the response of brain endothelial cells to oxidative stress. Cultured brain endothelial cells are pre-treated with acetaminophen and then exposed to the superoxide-generating compound menadione (25 microM). Cell survival, inflammatory protein expression, and anti-oxidant enzyme activity are measured. Menadione causes a significant (p<0.001) increase in endothelial cell death as well as an increase in RNA and protein levels of tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES. Menadione also evokes a significant (p<0.001) increase in the activity of the anti-oxidant enzyme superoxide dismutase (SOD). Pre-treatment of endothelial cell cultures with acetaminophen (25-100 microM) increases endothelial cell survival and inhibits menadione-induced expression of inflammatory proteins and SOD activity. In addition, we document, for the first time, that acetaminophen increases expression of the anti-apoptotic protein Bcl2. Suppressing Bcl2 with siRNA blocks the pro-survival effect of acetaminophen. These data show that acetaminophen has anti-oxidant and anti-inflammatory effects on the cerebrovasculature and suggest a heretofore unappreciated therapeutic potential for this drug in neurodegenerative diseases such as Alzheimer's disease that are characterized by oxidant and inflammatory stress.

Fig. 1. Acetaminophen increases endothelial cell survival

Brain endothelial cells (passages 7–10) were treated with acetaminophen (APAP) and cell survival was evaluated by MTT assay. In each experiment, the number of control cells i.e. viable cells not exposed to any treatment, was defined as 100%. (a) Endothelial cells were incubated with 0–300 µM of acetaminophen and cell survival measured. *p<0.05 vs. control; ***p<0.001 vs. control. (b) Brain endothelial cells were incubated with 100 µM APAP for 1 to 4 pretreatment periods. Each treatment period was 8 h. The number of control cells i.e. viable cells not exposed to any treatment, was defined as 100%. **p<0.01 vs. control; ***p<0.001 vs. control. Data are mean ± SD from 3 separate experiments.

Fig. 1. Acetaminophen increases endothelial cell survival

Brain endothelial cells (passages 7–10) were treated with acetaminophen (APAP) and cell survival was evaluated by MTT assay. In each experiment, the number of control cells i.e. viable cells not exposed to any treatment, was defined as 100%. (a) Endothelial cells were incubated with 0–300 µM of acetaminophen and cell survival measured. *p<0.05 vs. control; ***p<0.001 vs. control. (b) Brain endothelial cells were incubated with 100 µM APAP for 1 to 4 pretreatment periods. Each treatment period was 8 h. The number of control cells i.e. viable cells not exposed to any treatment, was defined as 100%. **p<0.01 vs. control; ***p<0.001 vs. control. Data are mean ± SD from 3 separate experiments.

Fig. 2. Endothelial cell survival in response to oxidative stress increases with acetaminophen pre-incubation

Brain endothelial cells were oxidatively challenged with 25 µM menadione for 3 h and cell viability was determined with MTT assay. The number of control cells i.e. viable cells not exposed to any treatment, was defined as 100%. (a) Brain endothelial cells were either pre-treated for 24 h () with acetaminophen (APAP) (0–300 µM) and then exposed to menadione or co-incubated () with APAP and menadione. *p<0.05 vs. menadione alone; ***p<0.001 vs. menadione alone. (b) Brain endothelial cells received 1 to 4 pretreatments of 100 µM acetaminophen (APAP) prior to 3 h exposure to menadione (25 µM). Each treatment period was 8 h. *p<0.05 vs. menadione alone; **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. (c) Rat brain endothelial cells were exposed to 4 pretreatments (32 h) of varying doses (10–100 µM) of acetaminophen (APAP) and then incubated with 25 µM of menadione for 3 h. ***p<0.001 vs. menadione alone. (d) Rat brain endothelial cells were exposed to 4 pretreatments (32 h) of varying doses (10–100 µM) of acetaminophen and then incubated with 1 mM H₂O₂ for 3 h. ***p<0.001 vs. H₂O₂ alone. Data are mean ± SD from 3 separate experiments.

Fig. 2. Endothelial cell survival in response to oxidative stress increases with acetaminophen pre-incubation

Brain endothelial cells were oxidatively challenged with 25 µM menadione for 3 h and cell viability was determined with MTT assay. The number of control cells i.e. viable cells not exposed to any treatment, was defined as 100%. (a) Brain endothelial cells were either pre-treated for 24 h () with acetaminophen (APAP) (0–300 µM) and then exposed to menadione or co-incubated () with APAP and menadione. *p<0.05 vs. menadione alone; ***p<0.001 vs. menadione alone. (b) Brain endothelial cells received 1 to 4 pretreatments of 100 µM acetaminophen (APAP) prior to 3 h exposure to menadione (25 µM). Each treatment period was 8 h. *p<0.05 vs. menadione alone; **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. (c) Rat brain endothelial cells were exposed to 4 pretreatments (32 h) of varying doses (10–100 µM) of acetaminophen (APAP) and then incubated with 25 µM of menadione for 3 h. ***p<0.001 vs. menadione alone. (d) Rat brain endothelial cells were exposed to 4 pretreatments (32 h) of varying doses (10–100 µM) of acetaminophen and then incubated with 1 mM H₂O₂ for 3 h. ***p<0.001 vs. H₂O₂ alone. Data are mean ± SD from 3 separate experiments.

Fig. 2. Endothelial cell survival in response to oxidative stress increases with acetaminophen pre-incubation

Brain endothelial cells were oxidatively challenged with 25 µM menadione for 3 h and cell viability was determined with MTT assay. The number of control cells i.e. viable cells not exposed to any treatment, was defined as 100%. (a) Brain endothelial cells were either pre-treated for 24 h () with acetaminophen (APAP) (0–300 µM) and then exposed to menadione or co-incubated () with APAP and menadione. *p<0.05 vs. menadione alone; ***p<0.001 vs. menadione alone. (b) Brain endothelial cells received 1 to 4 pretreatments of 100 µM acetaminophen (APAP) prior to 3 h exposure to menadione (25 µM). Each treatment period was 8 h. *p<0.05 vs. menadione alone; **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. (c) Rat brain endothelial cells were exposed to 4 pretreatments (32 h) of varying doses (10–100 µM) of acetaminophen (APAP) and then incubated with 25 µM of menadione for 3 h. ***p<0.001 vs. menadione alone. (d) Rat brain endothelial cells were exposed to 4 pretreatments (32 h) of varying doses (10–100 µM) of acetaminophen and then incubated with 1 mM H₂O₂ for 3 h. ***p<0.001 vs. H₂O₂ alone. Data are mean ± SD from 3 separate experiments.

Fig. 2. Endothelial cell survival in response to oxidative stress increases with acetaminophen pre-incubation

Brain endothelial cells were oxidatively challenged with 25 µM menadione for 3 h and cell viability was determined with MTT assay. The number of control cells i.e. viable cells not exposed to any treatment, was defined as 100%. (a) Brain endothelial cells were either pre-treated for 24 h () with acetaminophen (APAP) (0–300 µM) and then exposed to menadione or co-incubated () with APAP and menadione. *p<0.05 vs. menadione alone; ***p<0.001 vs. menadione alone. (b) Brain endothelial cells received 1 to 4 pretreatments of 100 µM acetaminophen (APAP) prior to 3 h exposure to menadione (25 µM). Each treatment period was 8 h. *p<0.05 vs. menadione alone; **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. (c) Rat brain endothelial cells were exposed to 4 pretreatments (32 h) of varying doses (10–100 µM) of acetaminophen (APAP) and then incubated with 25 µM of menadione for 3 h. ***p<0.001 vs. menadione alone. (d) Rat brain endothelial cells were exposed to 4 pretreatments (32 h) of varying doses (10–100 µM) of acetaminophen and then incubated with 1 mM H₂O₂ for 3 h. ***p<0.001 vs. H₂O₂ alone. Data are mean ± SD from 3 separate experiments.

Fig. 3. Oxidative stress-related increase in cytokine secretion is decreased by acetaminophen

Brain endothelial cells were pretreated (32 h) with acetaminophen (APAP) and incubated with 25 µM of menadione for 3 h. Supernatants of endothelial cell cultures were analyzed by ELISA for the presence of cytokines (a) TNFα, (b) IL-1α, and (c) IL-1β. *p<0.05 vs. menadione alone; **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. Data are mean ± SD values from 3 separate experiments.

Fig. 3. Oxidative stress-related increase in cytokine secretion is decreased by acetaminophen

Brain endothelial cells were pretreated (32 h) with acetaminophen (APAP) and incubated with 25 µM of menadione for 3 h. Supernatants of endothelial cell cultures were analyzed by ELISA for the presence of cytokines (a) TNFα, (b) IL-1α, and (c) IL-1β. *p<0.05 vs. menadione alone; **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. Data are mean ± SD values from 3 separate experiments.

Fig. 4. Oxidative stress-related increase in chemokine secretion is decreased by acetaminophen

Brain endothelial cells were pretreated (32 h) with acetaminophen (APAP) and incubated with 25 µM of menadione for 3 h. Supernatants of endothelial cell cultures were analyzed by ELISA for the presence of chemokines (a) MIP-1α and (b) RANTES. **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. Data are mean ± SD values from 3 separate experiments.

Fig. 4. Oxidative stress-related increase in chemokine secretion is decreased by acetaminophen

Brain endothelial cells were pretreated (32 h) with acetaminophen (APAP) and incubated with 25 µM of menadione for 3 h. Supernatants of endothelial cell cultures were analyzed by ELISA for the presence of chemokines (a) MIP-1α and (b) RANTES. **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. Data are mean ± SD values from 3 separate experiments.

Fig. 4. Oxidative stress-related increase in chemokine secretion is decreased by acetaminophen

Brain endothelial cells were pretreated (32 h) with acetaminophen (APAP) and incubated with 25 µM of menadione for 3 h. Supernatants of endothelial cell cultures were analyzed by ELISA for the presence of chemokines (a) MIP-1α and (b) RANTES. **p<0.01 vs. menadione alone; ***p<0.001 vs. menadione alone. Data are mean ± SD values from 3 separate experiments.

Fig. 5. Effect of acetaminophen on menadione-induced expression of cytokine and chemokine mRNA

Brain endothelial cells were pretreated (32 h) with varying concentrations (0–100 µM) acetaminophen (APAP) and incubated with 25 µM menadione for 3 h. Total RNA was extracted and RT-PCR performed with specific primers (MIP-1α, RANTES, IL-1α, IL-1β, TNFα) and PCR amplified products visualized (n=3). The housekeeping gene GAPDH was used to confirm loading equivalency between lanes. Data are mean ± SD from 3 separate experiments; a representative blot is shown

Fig. 5. Effect of acetaminophen on menadione-induced expression of cytokine and chemokine mRNA

Brain endothelial cells were pretreated (32 h) with varying concentrations (0–100 µM) acetaminophen (APAP) and incubated with 25 µM menadione for 3 h. Total RNA was extracted and RT-PCR performed with specific primers (MIP-1α, RANTES, IL-1α, IL-1β, TNFα) and PCR amplified products visualized (n=3). The housekeeping gene GAPDH was used to confirm loading equivalency between lanes. Data are mean ± SD from 3 separate experiments; a representative blot is shown

Fig. 6. Acetaminophen induces expression of Bcl2 in brain endothelial cells

Brain endothelial cells were pretreated (32 h) with acetaminophen (APAP) and incubated with 25 µM of menadione for 3 h. (a) Total RNA was extracted and RT-PCR performed with specific primers. PCR amplified products corresponding to Bcl2 were demonstrable as discrete bands at 331 bp. The housekeeping gene actin was used to confirm loading equivalency between lanes. Quantitation of densitometric scans from 3 separate experiments is shown in lower panel. ###p<0.001 vs. control; ^ap<0.001 vs. control; *p<0.05 vs. menadione alone; ***p<0.001 vs. menadione alone. (b) Total protein was extracted and western blots performed using antibodies specific for Bcl2. A western blot representative of 3 separate experiments is shown. Antibodies to GAPDH were used to confirm loading equivalency between lanes. Quantitation of densitometric scans from 3 separate experiments is shown in lower panel. ^ap<0.001 vs. control; ###p<0.001 vs. control; #p<0.05 vs. control; **p<0.01 vs. menadione alone;***p<0.001 vs. menadione alone. Data are mean ± SD from 3 separate experiments; a representative blot is shown

Fig. 7. Silencing Bcl2 expression blocks protective effect of acetaminophen

Brain endothelial cells were transfected with siRNA for Bcl2 or scrambled siRNA for 48 h. The cells were treated with 100µM of acetaminophen for 8 h and then exposed to 25 µM menadione for 3 h. Cell survival was assayed with MTT reagent. Survival in control (i.e. untreated) cultures was defined as 100%. #p<0.001 vs. control; ^ap<0.001 vs. Bcl2siRNA; ^bp<0.001 vs. menadione alone. Data are means ± SD values from 3 separate experiments. Inset: Bcl2 expression in endothelial cells transfected with siRNA for Bcl2, scrambled siRNA (neg control), or in non-transfected cells (cont).