Baicalin attenuates oxygen-glucose deprivation-induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase activation in PC12 cells

Abstract

Aim: To determine whether the flavonoid baicalin attenuates oxygen-glucose deprivation (OGD)-induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase (5-LOX) activation in PC12 cells.

Methods: The effects of baicalin and the 5-LOX inhibitor zileuton on the changes induced by OGD/recovery or H(2)O(2) (an exogenous reactive oxygen species [ROS]) in green fluorescent protein-5-LOX-transfected PC12 cells were compared.

Results: Both baicalin and zileuton attenuated OGD/recovery- and H(2)O(2)-induced injury and inhibited OGD/recovery-induced production of 5-LOX metabolites (cysteinyl leukotrienes) in a concentration-dependent manner. However, baicalin did not reduce baseline cysteinyl leukotriene levels. Baicalin also reduced OGD/recovery-induced ROS production and inhibited 5-LOX translocation to the nuclear envelope and p38 phosphorylation induced by OGD/recovery and H(2)O(2). In contrast, zileuton did not show these effects.

Conclusion: Baicalin can inhibit 5-LOX activation after ischemic injury, which may partly result from inhibition of the ROS/p38 mitogen-activated protein kinase pathway.

Figure 1

Effects of baicalin and zileuton on cell injury induced by OGD/recovery in PC12 cells. Cell viability was reduced after 2-h OGD and 24-h recovery in both GFP- and GFP-5-LOX-transfected cells. (A and B) Viability was significantly lower in GFP-5-LOX-transfected cells than in GFP-transfected cells. Baicalin and zileuton attenuated OGD/recovery-induced injury in both type cells in a concentration-dependent manner. (C) Cell death (necrosis) was evaluated by PI fluorescence staining in GFP-5-LOX-transfected PC12 cells (Scale bar=40 μm). (D and E) Baicalin and zileuton inhibited OGD/recovery-induced cell death in a concentration-dependent manner. Data are reported as mean±SD. n=15−17 (A and B) or 4 (D and E). ^bP<0.05, ^cP<0.01 vs corresponding control; ^eP<0.05, ^fP<0.01 vs OGD/recovery alone; ^hP<0.05, ⁱP<0.01 vs GFP-transfected cells.

Figure 2

Effects of baicalin and zileuton on 5-LOX translocation after OGD/recovery in GFP-5-LOX-transfected PC12 cells. (A) GFP-5-LOX was translocated into the nuclear envelope after 2-h OGD and 2-h recovery, which was inhibited by baicalin (1 μmol/L) but not by zileuton (1 μmol/L). Scale bar=40 μm. (B and C) Concentration-dependent results are summarized as mean±SD. n=6. ^bP<0.05, ^cP<0.01 vs OGD/recovery alone. ND, not detectable.

Figure 3

Effects of baicalin and zileuton on production of CysLTs after OGD/recovery in GFP-5-LOX-transfected PC12 cells. Baicalin (Ba, 10 μmol/L) reduced only the increased production whereas zileuton (Zi, 10 μmol/L) reduced both baseline and the increased production of CysLTs after OGD/recovery. Data are reported as mean±SD. n=4. ^bP<0.05, ^cP<0.01 vs control (no treatment). ^fP<0.01 vs OGD/recovery alone.

Figure 4

Effects of baicalin and zileuton on OGD/recovery-induced ROS production and H₂O₂-reduced viability in PC12 cells. (A and B) ROS production was determined after 2-h OGD and 0.5-h recovery in the absence or presence of baicalin and zileuton in wild-type PC12 cells. Baicalin, but not zileuton, inhibited ROS production. (C and D) Exposure to H₂O₂ (160 mol/L) for 24 h reduced cell viability in GFP-5-LOX-transfected PC12 cells. Over-expression of 5-LOX augmented H₂O₂- induced injury in PC12 cells. Baicalin (C) and zileuton (D) attenuated the reduced viability. Data are reported as mean±SD. n=8. ^bP<0.01, ^cP<0.01 vs control; ^eP<0.05, ^fP<0.01 vs OGD alone; ^hP<0.05, ⁱP<0.01 vs GFP-transfected cells.

Figure 5

Effects of baicalin and zileuton on H₂O₂-induced 5-LOX translocation in GFP-5-LOX-transfected PC12 cells. (A) H₂O₂ (160 μmol/L)–induced 5-LOX translocation was significantly inhibited by baicalin (1 μmol/L) but not by zileuton (1 μmol/L). Scale bar=20 μm. (B and C) Concentration-dependent results are reported as mean±SD. n=6−8. ^cP<0.01 vs H₂O₂ alone. ND, not detectable.

Figure 6

Effects of baicalin and zileuton on OGD/recovery- or H₂O₂-induced p38 phosphorylation in GFP-5-LOX transfected PC12 cells. Phosphorylation of p38 was inhibited by baicalin (1 and 10 μmol/L) but not by zileuton after 2-h OGD/1.5-h recovery (A) and exposure to H₂O₂ (160 μmol/L) for 20 min (B). Data are reported as mean±SD. n=4−6. ^bP<0.05, ^cP<0.01 vs corresponding control; ^eP<0.05, ^fP<0.01 vs OGD/recovery or H₂O₂ alone. ND, not detectable.