PYC prevented loss of SH-SY5Y cell viability. The viability was measured by the reduction of MTT. Acrolein caused loss of cell viability compared with control. 1 h pretreatment with PYC (50 and 100 μg/ml) and incubation for 24 h protected SH-SY5Y cells from acrolein cytotoxicity compared with group acrolein only. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only.

(A) PYC reduces ROS production in SH-SY5Y cells. ROS level was measured by the oxidation of DCFH-DA. Acrolein treatment increased ROS production in SH-SY5Y cells compared with control. 1 h pretreatment with PYC (50 and 100 μg/ml) and incubation for 24 h reduced ROS production by acrolein treatment in SH-SY5Y cell compared the culture treated with acrolein only. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only. PYC depletes O₂^·− production in SH-SY5Y cells. (B) O₂^·− level and (C) O₂^·− producing enzyme NADPH-oxidase activity was increased with acrolein treatment in SH-SY5Y cells compared with control. 1 h pretreatment with PYC (50 and 100 μg/ml) and incubation for 24 h reduced O₂^·− production by acrolein treatment in cell cultures compared with acrolein only. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p <0.05 and ^##p < 0.001 versus acrolein only.

PYC treatment protects SH-SY5Y cells from protein and lipid damages. Acrolein increased protein oxidation (protein carbonyl formation A & a), lipid peroxidation (4-HNE formation B & b), and protein nitration (3-NT formation C & c) in SH-SY5Y cells compared from acrolein induced protein damages compared with group acrolein only. Blot represents one sample of each group and each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only.

Cytosolic protein subunits of NADPH-oxidase p67^Phox (A & B), levels in the SH-SY5Y cells increased in membrane and declined in cytosol with acrolein exposure were significantly attenuated by PYC (50 μg/ml). In same fashion p47^Phox (C), and p407^Phox (D) also show increase with acrolein exposre and significantly decreased with PYC treatment. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only.

Schematic presentation of proposed mechanism of protective effects of pycnogenal (PYC) on acrolein-induced cytotoxicity in SH-SY5Y cells. The thiol (-SH) – reactive property of acrolein activates NADPH-oxidase complex, leading to the production of superoxide (O₂^−.). Normally superoxide dismutase (SOD) converts O₂^−. into hydrogen peroxide (H₂O₂). O₂^−. activates nitric oxide synthase (iNOS) leading to the production of nitric oxide (NO) and the formation of peroxynitrites (ONOO⁻), which ultimately causes oxidative/nitrosative stress. Increased oxidative/nitrosative stress causes mitochondrial dysfunction, lipid peroxidation, protein and DNA damage, which can result in cell death. Lipid peroxidation further produces toxic aldehyde products including elevated acrolein that further aggravates degenerative process. ONOO⁻ is known to activate poly(ADP-ribrosyl) polymerase (PARP) leading to ATP depletion and cell death. PYC prevents assembling of NADPH-oxidase subunits (both in cytosol and the membrane), and has protective effects on the beginning of acrolein-induced deleterious cascade. PYC prevents O₂^−. production from NADPH-oxidase activation and NO from iNOS. PYC scavenges O₂^−., NO, ONOO⁻, H₂O₂ and hydroxyl radicals (OH^.) to prevent oxidative/nitosative stress, and elevates internal antioxidant glutathione (GSH) defenses that can enhance cell survival.

Phase contrast microphotographs of SH-SY5Y cells treated with acrolein with and without PYC. Acrolein treatment resulted in significant cell death with some membrane blobbing, but no other morphological changes in surviving SH-SY5Y cells. PYC (50 and 100μg/ml) significantly has reduced cell death, even at high acrolein levels. (A) Control; (B) treated with acrolein 1 μM; (C) acrolein 5 μM; (D) acrolein 10 μM; (E) acrolein 25 μM; (F) treated with PYC 50 μg/ml; (G) acrolein 1 μM + PYC 50 μg/ml; (H) acrolein 5 μM + PYC 50 μg/ml; (I) acrolein 10 μM + PYC 50 μg/ml; (J) acrolein 25 μM + PYC 50 μg/ml; (K) treated with PYC 100 μg/ml; (L) acrolein 1 μM + PYC 100 μg/ml; (M) acrolein 5 μM + PYC 100 μg/ml; (N) acrolein 10 μM + PYC 100 μg/ml; (O) acrolein 25 μM + PYC 100 μg/ml. Microphotographs were taken at 40 X and calibration bar was 10μ.

Level of GSH system was alleviated with PYC treatment in SH-SY5Y cells. Acrolein caused loss of GSH (A) increased GSSG (B) and discrepancy of GSH/GSSG (C) in SH-SY5Y cells compared with control. 1 h pretreatment with PYC (50 and 100 μg/ml) and incubation for 24 h protected cells from acrolein induced adverse effect on GSH system compared with group acrolein only. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only.

NADPH-oxidase subunit proteins in SH-SY5Y cells were significantly affected with acrolein exposure. Cells were processed for immunobloting followed by Western-blot. The levls of p67^Phox (B), p47^Phox (C), and p40^Phox (D) demonstrated increase in membrane and p67^Phox decreased in cytosol (G). The effect of acrolein exposure was attenuated with PYC (50 μg/ml ). The gp91^Phox (A) and p22^Phox (E) failed to demonstrate significant changes. Na⁺/K⁺-ATPase (F) and GAPDH (G) show equal proteins.

Membrane protein subunits of NADPH-oxidase gp91^Phox (A) and p22^Phox (B) show non significant elevation with acrolien exposure. PYC attenuates the levels of both subunits in membrane.