| OBJECTIVE:More recently, stroke has become one of the major causes of death worldwide. Intravenous thrombolytic therapy is one proven effective treatment in the acute setting. However, reperfusion therapy is a double-edged sword:on one hand, it can rescue ischemic brain tissue and improve clinical symptom; on the other hand, some patient condition become more deteriorating than before, it is known as cerebral ischemia/reperfusion injury. While it is a very complicated process, scientists are exploring the reperfusion injury mechanism for the treatment of brain diseases.Programmed cell death 4 was identified as a new tumor suppressor gene. PDCD4 inhibits translation initiation mRNA-specifically by interacting with translation initiation factor eIF4A and competing with eIF4Gc for binding to eIF4A, which leads to cellular cleavage and proliferation markedly inhibited. In the meantime, studies show that PDCD4 could exacerbate inflammation. While the mechanism of PDCD4 in many kinds of tumor has been studied more thoroughly, the role of PDCD4 in cerebrovascular diseases is not clear. Therefore, in the present study, we used an in vivo middle cerebral artery occlusion (MCAO) model and in vitro cell cultures by oxygen glucose deprivation (OGD) for the first time to investigate the expression and function of PDCD4 in ischemic brains.METHODS:1. The function and expression of PDCD4 in mice brain after MCAO1.1 Wild tipe and PDCD4-/- mice were randomly divided into different groups. the ischemia model was induced by occlusion of the middle cerebral artery (MCAO) with a microfilament. Neurological scoring, TTC staining, HE staining and TUNEL staining were used to evaluate the damage to central nervous system.1.2 The expression of PDCD4 was detected by Western blot, while the distribution was detected by Immunofluorescence staining.2. The mechanism of ischemia/reperfusion injury mediated by PDCD4 pathway2.1 TUNEL staining was performed to detect neuron apoptosis; besides, The expression of Bcl-2/Bax and caspase-3 were examined by Western blot.2.2 To provide evidence to establish the relationship between PDCD4 expression and immune response in the brain, relative levels of inflammatory factors IL-6, IL-10, MCP-1, IL-1β, TNF-α were detected by Real-time RT PCR.2.3 The autophagosomes were observed by transmission electron microscope. Autophagy related protein LC3B, Beclin-1 and Atg5 were examined by Western blot, which were to determine the level of autophagy. BIP and CHOP were also examined by Western blot in order to detect the level of endoplasmic reticulum stress.3. The expression of PDCD4 in different cells after OGD3.1 siRNA-PDCD4 was used to knock down PDCD4, while pEFGP-PDCD4 was to overexpress PDCD4. After transfection, PC-12 and BV-2 cells were subjected to OGD treatment. The expressions of PDCD4, LC3B, p-ERKl/2, BIP, CHOP were detected by Western blot.3.2 U0126 was used to inhibit MAPK/ERK signaling pathway in OGD/Reperfusion injury. The results will show the role of MAPK/ERK in ERS and autophagy induction. We also analyzed the levels of inflammatory cytokines in BV-2 lines by Real-time RT PCR.RESULTS:1. PDCD4 deficiency ameliorated stroke outcomeUsing TTC staining, we observed the reduced infarction volume in PDCD4-/-ischemic mice, which was consistent with neurologic scoring. HE staining, Nissl staining and TUNEL further indicated that PDCD4 deficiency protected against ischemia-induced neuron injury and death.2. PDCD4 expression was decreased at first and then increased after MCAO2.1 Western blot was used to analyze the expression of PDCD4 after MCAO. We found that PDCD4 expression in the ischemic penumbra was markedly decreased at the early period, but it had a high expression at 48h after cerebral ischemia reperfusion.2.2 We found that PDCD4 was mainly expressed in neurons and microglia cells by confocal immunofluorescence staining.2.3 PDCD4 expression in PC-12 showed a similar result as in vivo, but PDCD4 expression had been rising in BV-2 cells after OGD/Reperfusion.3. PDCD4 could increased the level of apoptosis significantlyTUNEL staining was used to assess the apoptotic like neurons; a significant number of TUNEL-positive neurons manifested as brownish staining in the nuclei in WT mice. At the same time, Bcl-2/Bax and caspase-3 was tested to identify the level of apoptosis by western blot.4. PDCD4 has a profound effect on the inflammatory response after reperfusion4.1 We examined the expression of cytokines in ischemic brains from both WT and PDCD4-/- mice. It was found that PDCD4-/- mice expressed significantly less cytokines including IL-6, MCP-1 and TNF-a.4.2 In vitro, we got the same results with in vivo.5. PDCD4 negatively regulated autophagy5.1 The number of typical autophagosomes with double membranes was increased in PDCD4-/- mice compared with WT mice after MCAO when observed with transmission electron microscopy. Beclin-1 was not affected by either overexpressed or silenced PDCD4 under autophagy-inducing conditions, indicating that PDCD4 inhibits autophagy in a Beclin-1 independent manner. However, we found that that forced PDCD4 expression remarkably inhibited formation of the Atg12-Atg5 complex and free Atg5.5.2 BIP protein level was markedly enhanced in the brain from PDCD4-/-ischemia mice, while CHOP took on a descenting trend.5.3 MAPK/ERK signaling took part in the PDCD4-mediated pathway.5.4 In vitro, BIP and LC3B were both decreased when MAPK/ERK signaling pathway was blocked.CONCLUSION:1. PDCD4 exacerbates stroke outcomes.2. The expression of PDCD4 is different in nerve cells after OGD treatment. Expression of PDCD4 in PC-12 decreased firstly and then increased, while PDCD4 was upregulated all the time in microglias.3. PDCD4 could significantly promote the apoptosis of neurons and inhibit the level of autophagy, and it could also enhance the damage of brain tissue by inflammatory reaction. |
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