Effects Of Gadd45b In The Pathological Process Of Brain Ischemia And On Brian Plasticity

Aims: Growth arrest and DNA-damage inducible protein beta(Gadd45b) belongs to the Gadd45 family, and is demonstrated related to neuronal activity and neurogenesis. Rat brain ischemia and reperfusion injury induces the protein and mRNA expression of Gadd45 b. It mediated neuron apoptosis and may stimulate axon plasticity and recovery after stroke. Autophagic cell death, also called type II programmed cell death, is involved in the pathology of brain ischemia and reperfusion. However, little is known of the effect of Gadd45 b on autophagy. Moreover, Gadd45 b increased the expression of brain derived neurotrophic factor(BDNF), related to axon plasticity and stimulates recovery in cerebral ischemia. The ubiquitin-proteasome system(UPS) is involved in the degradation of intracellular protein. However, little is known of the molecular mechanisms of how Gadd45 b expression regulated in brain ischemia. The aim of this study is to investigate the effect of Gadd45 b on autophagy and the possible mechanism in the pathology of cerebral ischemia/reperfusion injury, and to analyze the effect of Huwe1 on Gadd45 b and brain plasticity.Methods: We used oxygen-glucose deprivation and reperfusion(OGD/R) model of rat primary cortex neuron cell, which was OGD 3 h and reperfusion 0 h,6 h, 24 h,48 h,72 h and 120 h. CCK-8 and LDH assay were used to examine the cortex neuron cell viability and injury. We employed lentivirus shRNA-Gadd45 b to inhibit the Gadd45 b expression, and examined the level of autopahy-related molecular(ATG5, LC3, Beclin-1, ATG7, ATG3), apoptosis molecular(Bax, Bcl-2, Cleaved caspase3, p53) and JNK/p38 pathway by western blotting. In addition, we examined the cortex neuron cell apoptosis by TUNEL after co-treatment with autophay inhibitor or JNK/p38 pathway inhibitor. Neuron neurites injury was assayed by double immunofluorescent labeling with Tuj1 and LC3 B. We also employed lentivirus shRNA-Huwe1 to silence the Gadd45 b expression, and examined the interaction with Huwe1 and Gadd45 b by CO-IP assay. CHX was used to examine the effect of Huwe1 on Gadd45 b protein stability. The BSP assay was used to examine the effect of Huwe1 or Gadd45 b on BDNF IXa methylation. The effect of Huwe1 or Gadd45 b on BDNF, receptors TrkB, p75 NTR and downstream effectors was examined by western blot.Results: Under OGD/R, the neuronal cell viability declined progressively as the reperfusion time prolonged. The level of Gadd45 b was on peak at 24 h after OGD treatment. Lentivirus shRNA-Gadd45 b obviously decreased the expression of Gadd45 b at 24 h after OGD treatment, increased the expression of autopahy-related molecular(Beclin-1, ATG5, ATG7 and ATG3), the rate of LC3 II/LC3 I and apoptosis protein Bax and Cleaved caspase3, but decreased the anti-apoptosis protein Bcl-2 expression. Moreover, co-treatment with shRNA-Gadd45 b and autophay inhibitor 3-MA or Wortmannin partly decreased the ratio of LC3-II/LC3-I and mildly decreased the apoptosis. Silencing Gadd45 b expression decreased the level of autophay-related pathway p-p38 and apoptosis-related pathway p-JNK. Co-treatment with shRNA-Gadd45 b and p38 pathway inhibitor obviously increased the apoptosis. On the contrary, co-treatment with shRNA-Gadd45 b and JNK pathway inhibitor obviously decreased the apoptosis. Inhibition of Huwe1 obviously increased the expression of Gadd45 b and BDNF at 24 h after OGD. CO-IP results showed that there existed the interaction with Huwe1 and Gadd45 b.Treatment with CHX inhibited the endogenous expression of Gadd45 b, but increased the expression of Gadd45 b after co-treated with lentivirus shRNA-Huwe1. The shRNA-Gadd45 b decreased the levels of BDNF and downstream, while shRNA-Huwe1 increased it. In addition, the shRNA-Huwe1 decreased the methylation of the fifth CpG islands, and shRNA-Gadd45 b treatment increased the methylation of the forth CpG islands.Conclusions: Gadd45 b regulated autophay and apoptosis. The expression of Gadd45 b was regulated by Huwe1 in the pathology of cortex neuron cell OGD/R. Gadd45 b as well as Huwe1 mediated the methylation of BDNF IXa. It may provide a novel route for neurons following brain ischemia-reperfusion injury and a new support for clinical treatment.