HO-1 Signaling Activation By Pterostilbene Treatment Prevents Cerebral Ischemia Reperfusion Injury

Ischemic stoke is one of most threatening disease to human health. Recently, cerebral ischemia reperfusion(IR) injury theory is fully developed,but therapeutic methods have no breakthrough. Thrombolytic agents still are the main application used in the ischemic stroke. Therefore, alternative agents that prevent ischemia injury have been intensely explored in neuron science. Pterostilbene(PTE), a natural dimethylated analog of resveratrol from blueberries, has greater lipophilicity and a higher potential bioavailability for cellular uptake than resveratrol which is known to have diverse pharmacological activities, including anti-inflammation, anti-oxidation, anti-apoptotic and analgesic properties. HO-1 is a stress response anti-oxidative protein, which is highly inducible in response to cerebral ischemia stimulation. PTE has been shown to be beneficial for some nervous system, cardiovascular, metabolic and hepatic diseases. Studies have also shown that resveratrol can attenuate cerebral IR injury via multiple mechanisms. However, the effects of PTE on cerebral IR injury and the mechanisms responsible for these effects have not been elucidated. Above all, this study was designed to evaluate the protective effect of PTE on cerebral IR injury and to investigate its potential effects on HO-1 signaling with murine hippocampal neuronal HT22 cells simulated ischemia reperfusion(SIR) model and mouse global cerebral IR model.Experiment 1: HO-1 signaling in PTE treatment prevents IR injury in murine hippocampal neuronal HT22 cells SIR model.Objective: To investigate HO-1 signaling in cerebral protection of PTE treatment with HT22 cells SIR model.Methods: HT22 cells were divided into 4 groups(control, PTE 1.25μM, PTE 2.5μM, PTE 5μM), cell viability, LDH leakage and HO-1 expression were detected to evaluate the effects of PTE treatment on HT22 cells(n=8).The HT22 cells were randomly divided into 4 groups(SIR, PTE 1.25μM+SIR, PTE 2.5μM+SIR, or PTE 5μM+SIR), cell viability and LDH leakage were tested, TUNEL dectected cell apoptosis, Western blot analysis HO-1,NAD(P)H:quinone oxidoreductase 1(NQO1) and glutathione S-transferases(GST) protein expression(n=8).HO-1 small interfering RNA(si RNA) was to block HO-1 expression, HT22 cells were randomly divided into 4 groups: control si RNA+SIR, control si RNA+PTE+SIR, HO-1 si RNA+PTE+SIR and HO-1 si RNA+SIR. Cell viability and LDH leakage were dectected, Western blot analysis HO-1, NQO1, GST protein expression(n=8).Results: PTE had no poison effect on HT22 cells, but up-regulated HO-1 expression in a dose-dependent manner(P<0.05). PTE treatment increased cell viability, decreased LDH leakage and cell apoptosis, and up-regulated HO-1, NQO1 and GST expression(P<0.05). While HO-1 si RNA suppressed HO-1 expression, it reversed the effect of PTE treatment on cell viability, LDH leakage, HO-1, NQO1 and GST expression in HT22 cells SIR model(P<0.05).Conclusion: PTE treatment prevents HT22 cells SIR model injury via HO-1 signaling in vitro.Experiment 2: HO-1 signaling in PTE treatment prevents IR injury in mouse global cerebral IR model.Objective: To evaluate HO-1 signaling in cerebral protection of PTE treatment with mouse global cerebral IR model.Methods: 32 mice were randomly divided into 4 groups(control, PTE 2.5mg/kg, PTE 5mg/kg, PTE 10mg/kg), neurological scorings, brain edema, and ion content, and HO-1 expression were detected to evaluate the effects of PTE treatment on mice. The 32 mice were randomly divided into 4 groups(SIR, PTE 2.5mg/kg+IR, PTE 5mg/kg+IR, PTE 10mg/kg+IR), after 24 h cerebral IR, neurological scorings, brain edema, and ion content were tested, Neu N marked viable neurons, TUNEL dectected cell apoptosis, Western blot analysis HO-1, NQO1 and GST protein expression. Zinc protoporphyrin(Zn PP) was to block HO-1 expression, 32 mice were randomly divided into 4 groups: IR, PTE+IR, Zn PP+PTE+IR and Zn PP+IR. Neurological scorings, brain edema, and ion content were dectected, and Western blot analysis HO-1, NQO1, GST protein expression.Results: PTE had no effect on neurological scorings, brain edema, and ion content of mice, but up-regulated HO-1 expression in a dose-dependent manner(P<0.05). PTE treatment increased neurological scorings and neurons viability, decreased brain edema, ion content and cell apoptosis, also up-regulated HO-1, NQO1 and GST expression(P<0.05). While Zn PP blocked HO-1 expression, it reversed the effect of PTE treatment on neurological scorings, brain edema, ion content, HO-1, NQO1 and GST expression in mouse global cerebral IR model(P<0.05).Conclusion: PTE treatment prevents mouse global cerebral IR model injury via HO-1 signaling in vivo.