to H2O2, there was a significant increase in the phosphorylation of ASK1 compared with the control group. Pretreatment with either 50 or 400 mM 20E for 24 h before H2O2 exposure inhibited the phosphorylation of ASK1 induced by H2O2. Total ASK1 did not change in any group. The results were similar in the cases of MKK4/7 and JNK. 20E protects rats against transient focal cerebral ischemic injury To further assess whether 20E had neuroprotective effects against cerebral ischemia in vivo, a model of transient focal ischemia produced by MCAO was employed. MCAO for 2 h, followed by a 24-h reperfusion, resulted in a large ipsilateral cerebral infarction, which was shown by a white region after TTC staining at 24 h of reperfusion. An intraperitoneal injection of 20E at the onset of reperfusion significantly reduced the cerebral infarction area induced by MCAO. 9 20-Hydroxyecdysone Inhibits Cerebral Injury 20E attenuates antioxidant potential descent and lipid peroxidation in the rat cortex induced by transient focal cerebral ischemia To evaluate oxidative damage following cerebral ischemia and the anti-oxidative effects of 20E in rats, we measured T-AOC, SOD activity, GPX activity level and MDA level in the rat cortex. As shown in Fig. 9CE, the T-AOC, SOD activity and GPX activity level in the rat ischemic cortex were reduced from 3.6260.31 to 1.5560.3 mmol/mg protein, 186.4465.21 to 129.9966.75 U/mg protein and 24.4263.04 to 14.0662.37 mU/mg protein, respectively. However, 20E dramatically increased the T-AOC, SOD activity and GPX activity level to 2.1260.24 or 3.5860.3 mmol/mg protein, 146.88611.18 or 176.2968.84 U/mg protein and 17.4963.66 or 20.8362.97 mU/mg protein, respectively. In addition, rat focal ischemia also caused an increase in the levels of MDA in ischemic cortex from 5.5160.54 to 12.7561.3 nmol/mg protein, which was 
dramatically decreased by 20E to 11.0960.71 and 8.360.72 nmol/mg protein, respectively. Discussion Oxidative stress is a major cause of neuronal injuries induced by cerebral ischemia. H2O2 has been extensively used as an inducer of oxidative stress. Thus, we used LY341495 H2O2-induced injury in B35 neuroblastoma cells in vitro to simulate the oxidative damage following cerebral ischemia, attempting to search for a naturally occurring drug with neuroprotective effects. 20E, an insect steroid hormone, exhibits a potent antioxidative effect. This study demonstrates that 20E reversed the H2O2-induced cell injury, which was characterized by a decrease in cell viability, release of LDH, and mitochondrial membrane potential dissipation and increases in the number of TUNEL-positive and cleaved caspase-3-positive cells. Four specific actions by 20E may contribute to this neuroprotective effect: first, 20E inhibits the generation of ROS and restores cellular antioxidant potential; second, 20E blocks oxidative stress-induced increases of i; third, 20E reduces the production of NO and the expression of iNOS protein, which may be mediated by inhibition of NF-kB activation; fourth, 20E suppresses the activation of the ASK1MKK4/7-JNK stress signaling pathway induced by oxidative stress. Finally, we establish a rat MCAO model to further confirm the neuroprotective effect of 20E. The results of our in vivo studies showed that 20E significantly attenuated ischemia injury caused by MCAO in rats. These findings indicate that 20E is useful as a potential protective agent against cerebral ischemic injury. Ischemia could increase production of ROS