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Figure legends 509
Fig 1. Chemical structure of isochaihulactone.
510 511
Fig 2. Attenuation of H2O2 -induced injury cell by isochaihulactone in neuronally 512
differentiated PC12 cells (nPC12). isochaihulactone or 100 µM α-tocopherol was added to the 513
cultures 3 h before the addition of H2O2. Cells were incubated with 200 µM H2O2 for 24 h for 514
MTT, LDH or apoptosis assay. Pretreatment with isochaihulactone protected nPC12 cells 515
against H2O2-induced injury by increasing cell viability (A) and decreasing H2O2-induced 516
cytotoxicity. The 100 µM α-tocopherol was used as a positive control (PC). (B). In addition, 517
isochaihulactone (10 µM) pretreatment decreased DNA fragmentation (C), chromatin 518
condensation (D) Caspase-3 and PARP cleavage (E), apoptotic characteristics induced by 519
H2O2. Data are presented as mean ± standard deviation (SD) (n = 3). aP < 0.05 as compared to 520
control group; bP < 0.05 as compared to H2O2 treated group.
521 522
Fig 3. Effect of isochaihulactone on H2O2-induced intracellular accumulation of reactive 523
oxygen species (ROS) and lipid peroxidation and downregulation of antioxidant enzyme 524
(SOD and GPx) activity in neuronally differentiated PC12 cells (nPC12). Pretreatment with 525
isochaihulactone attenuated the H2O2-induced accumulation of ROS (A) and lipid 526
peroxidation (B). In addition, isochaihulactone (10 µM) pretreatment maintained the activity 527
of SOD (C) and GPx (D) as controls. Isochaihulactone also rescued mRNA transcription of 528
SOD1 and SOD2, which was inhibited by H2O2 (E). Data are presented as mean ± standard 529
deviation (SD) (n = 3). aP < 0.05 as compared to control group; bP < 0.05 as compared to H2O2
530
treated group.
531 532
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Fig 4. Modulation of the cyclooxygenase 2 (COX-2) isozyme and NF-kappa B subunits 533
(RELA and P50) by isochaihulactone pretreatment in H2O2-treated neuronal PC12 cells 534
(nPC12). Treatment with H2O2 induced mRNA expression of COX-2, but not of COX-1, and 535
isochaihulactone pretreatment decreased this mRNA increase (A). Isochaihulactone 536
pretreatment also decreased COX-2 protein expression induced by H2O2 (B). In addition, 537
pretreatment with isochaihulactone decreased the mRNA expression of RELA and P50 (C).
538
Data are presented as mean ± standard deviation (SD) (n = 3). Relation to control in (A) to (C) 539
is relative to untreated control group.
540 541
Fig 5. Effect of isochaihulactone on plasma MDA level and SOD and GPx activities in 542
D-galactose–treated (aged) mice. The control group received subcutaneous (s.c.) injections of 543
phosphate-buffered saline. The aged group received D-galactose (100 mg/kg, s.c.). The 544
isochaihulactone group received D-galactose (100 mg/kg/day, s.c.) plus isochaihulactone (10 545
mg/kg/day, s.c). Treatments were administered for 6 weeks. Isochaihulactone treatment 546
attenuated the aging characteristics of increased MDA level and downregulated SOD and GPx 547
activities. In addition, neuronal damage analysis. H&E staining shows that pyknotic nucleis in 548
galactose-treated group (middle) were significantly increased compared with vehicle-treated 549
group (left) and decreased in galactose + isochaihulactone treated group (right) compared with 550
galactose alone group in the CA1 subfield of hippocampus after 6 weeks of administration (D).
551
Data are presented as mean ± standard deviation (SD) (n = 3 mice). aP < 0.05 as compared to 552
control group; bP < 0.05 as compared to H2O2 treated group.
553
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