| Background:The ischemic cerebral stroke is one of the most serious health-threaten diseases in the world, with the characteristics of high incidence and high morbidity and fatality rate. The common treatment is mainly focused on achieving the early-reperfusion of ischemic tissue, while there is an obvious limitation about this treatment. On one hand, the early-reperfusion could recover the function of ischemic tissue and relief the disease; on the other hand, the reperfusion could cause the ischemia/reperfusion injury, worsen the disease. Besides the cerebral stroke, liver ischemia, kidney ischemia and myocardial infarctionalso could cause the ischemia/reperfusion injury.The pathogenesis of ischemia/reperfusion injury is multi-factorial and unclear, although a growing number of studies have found the oxidative stress, inflammation and intracellular calcium release do the trick for the ischemic cerebral injury, and we still lack the ideal neuroprotective drugs to intervene this injury, which make the ischemic diseases one of worse prognosis diseases in the world. Meanwhile, the potential role of epigenetic modification is getting more and more attention.Recently, studies have highlighted the importance of epigenetic mechanisms such as histone modifications, DNA methylation, chromatin remodeling and non-coding RNAs (ncRNAs) in the pathogenesis of stroke, which orchestrate almost every aspect of central nervous system. Among them, histone deacetylases (HDACs)-mediated epigenetic mechanisms play important roles in the homeostasis of histone acetylation and gene transcription. There are four major HDACs classes which are classified based on their structures and expression patterns. Class Ⅰ (HDACs 1,2,3, and 8), class Ⅱ (HDACs 4,5,6,7,9, and 10) and class Ⅳ (HDAC 11) are Zn2+-dependent for enzymatic activity; whereas the class Ⅲ-Sirtuins (SIRT1-7) are NAD+-dependent. Each subtype of HDACs exerts its specific biological function in thephysiological activities.Although HDAC inhibitors have neuroprotective properties in animal models for various neurological diseases including Alzheimer’s disease and ischemic stroke, current HDAC inhibitors are mostly nonselective. While different HDACs serve very different functions, using the pan-selected HDACis could cause serious adverse reaction. Therefore, it is necessary to elucidate the functional role of individual HDACs in ischemic stroke. Our previous studies have characterized the expression patterns of individual HDACs in rats after cerebral ischemia/reperfusion injury. We found that among Zn2+-dependent HDACs, HDAC4 and HDAC5 were markedly decreased in ischemic strokeand demonstrated that HDAC4 and HDAC5 protected cells from death through reducing HMGB1 expression and release. Interestingly, we have observed that HDAC9 was significantly upregulated in the ischemic brain. However, the role of HDAC9 in ischemic stroke keeps unknown, and it’s necessary to design more effective and specific HDAC inhibitors in ischemic stroke treatment.Dysfunction of the vascular endothelium plays a central role in the pathogenesis of ischemic diseases. Brain endothelial cells are the structural basis of the blood brain barrier (BBB), which serves an important role on the internal environment homeostasis. In the ischemic cerebral injury, endothelial cells trigger the inflammation, increase the cell permeability and induce the cell apoptosis, worsen the injury. In the CNS, autophagy is triggered as an adaptive mechanism by various stressors, including cerebral ischemia, nutrient deprivation or excitotoxic stimuli. Epigenetic modification could regulate the autophagy. The triggered autoghagy provides nutrients and eliminates the damaged organelles to promote cell survival, but others also report that autophagy can cause autophagic death to aggravate the ischemiaoutcome. It is necessary to further explore the role and mechanism of endothelial cells autophagy in ischemic diseases.In conclusion, our study for the first time clarifies the important role of HDAC9 in the ischemic/reperfusion injury in vivo and in vitro. The HDAC9-mediated autophagy plays a crucial role in the endothelium injury. Meanwhile, to make our conclusions more reasonable, we also construct the liver ischemic/reperfusion injury model. Using another ischemic model, we obtained the consistent conclusion.Objective:1. Investigate the expression changes of HDAC9 and its potential role in rat cerebral ischemic/reperfusion injury.2. Discuss the role of HDAC9 in the endothelium dysfunction under OGD condition.3. Discuss the role of HDAC9-mediated autophagy in the endothelial dysfunction after OGD injury.4. Investigate the expression changes of HDAC9 and its potential role in rat liver ischemic/reperfusion injury.Methods:Part Ⅰ:The expression change and fuction of HDAC9 in the cerebral ischemic/reperfusion injury1.1 The expression changes of HDAC9 in the cerebral cortex after cerebral I/RI1.1.1 To build the MCAO model in Sprague-Dawley ratsThe Sprague-Dawley rats (250-280g) were randomly divided into two groups, the sham group and the MCAO group. After the operation, TTC staining analysis and neurological scoring system were used to assess the model.1.1.2 The expression distribution of HDAC9 in the cerebral cortexThe expression distribution of HDAC9 was detected by the RT-PCR, Western Blot and immunofluorescence analysis.1.1.3 The expression changes of HDAC9 in the cerebral cortex after I/RIThe expression changes of HDAC9 after cerebral I/RI were detected by Real time RT-PCR and Western Blot analysis respectively.1.2 HDAC9 gene silencing via the intraventricular lentivirus injection using the stereotaxis instrument1.2.1 To build the HDAC9 gene silencing rat modelConstruct the shRNA-HDAC9 lentivirus vector. Using the stereotaxis instrument to intraventricular inject the shRNA-HDAC9 lentivirus.1.2.2 After the surgery, Western Blot and immunofluorescence analysis were used to detect the HDAC9 gene silence efficiency.1.3 The potential role and mechanism of HDAC9 gene silencing on the ischemic stroke outcomes1.3.1 The function of HDAC9 gene silencing on the ischemic stroke outcomesAfter successful HDAC9 gene silencing, randomly divided the rats into four groups and induced ischemia in the left hemisphere by MCAO 1 week after the HDAC9 gene silencing.48 hour after the reperfusion, sacrifice the rat. Detect the function of HDAC9 gene silencing on stroke outcomes via TTC staining, cerebral edema calculation and neurologic deficit scores analysis.1.3.2 The potential mechanism of HDAC9 gene silencing on the ischemic stroke1.3.2.1 48 hour after the reperfusion, sacrifice the rat. Using evans blue staining detects the BBB permeability after HDAC9 gene silencing. Meanwhile, transmission electron microscopy analysis was also used to detect the injury after MCAO.1.3.2.2 48 hour after the reperfusion, sacrifice the rat. Western Blot was used to detect the expression changes of tight junction proteins (ZO-1, Occludin, Claudin-5) after HDAC9 gene silencing.Part Ⅱ:The role of HDAC9 on endothelial dysfunction under OGD condition2.1 The Oxygen and Glucose Deprivation (OGD) model2.1.1 Primary Brain Microvessel Endothelial Cells (BMVECs) cultureWe used the rat primary BMVECs for study.2.1.2 The expression changes of HDAC9 in the BMVECs after OGD injuryPlant the BMVECs on the culture dish, divided the cells into two groups randomly, the control group and the OGD group. After different reoxygenation time, harvest the cells, using Western Blot to detect the expression changes of HDAC9.2.2 The role of HDAC9 on BMVECs dysfunction after OGD treatment2.2.1 After transfect the BMVECs with shRNA-HDAC9 or pGV230-HDAC9 overexpression plasmid, detect the transfection efficiency via Western Blot.2.2.2 After the successful transfection, divided the cells randomly and stimulated individually. Detect the inflammation factors via Real time RT-PCR and the cells apoptosis via flow cytometric analysis under OGD treatment.2.3 The role of HDAC9 on BMVECs permeability changes after OGD treatment2.3.1 Cell permeability assay in vitro:Divided the cells randomly, after the successful transfection and OGD treatment, we used the cell permeability assay to detect the cell permeability.2.3.2 Divided the cells randomly, after successful transfection and OGD treatment, Western Blot was used to detect the expression changes of tight junction proteins (ZO-1, Occludin, Claudin-5) after HDAC9 gene silencing. Part Ⅲ:The functional role of HDAC9-mediated autophagy on BMVECs injury under OGD condition3.1 The role of HDAC9 on BMVECs autophagyBMVECs were divided randomly after successful transfection. Western Blotwas used to detect the changes of autophagic related proteins after OGD treatment. Meanwhile, Real time RT-PCR was used to detect the changes of p62 mRNA. Electron microscope analysis was used to detect the typical autophagosomes with double membranes in BMVECs after OGD treatment.3.2 The potential role of autophagy on BMVECs under OGD conditionThe classical mTOR inhibitor-rapamycin was added to activate the autophagy. Cell permeability assay was used to detect the BMVECs permeability. Meanwhile, Real time RT-PCR was used to detect the inflammation factors.Part Ⅳ:The expression change and fuction of HDAC9 in the liver ischemic/reperfusion injury4.1 The expression changes of HDAC9 in liver I/RI4.1.1 To build the rat liver I/R modelThe Sprague-Dawley rats (250-280g) were randomly divided into two groups, the sham group and the liver I/R group. After the operation, HE staining analysis was used to assess the model.4.1.2 The expression changes of HDAC9 after liver I/RIThe expression changes of HDAC9 after liver I/RI were detected by the immunohistochemistry staining and Western Blot analysis.4.2 The changes of HDAC9 on liver sinusoidal endothelial cells (LSECs) after OGD treatment4.2.1 To culture the LSECsIn our study, we use the RC-RM-0037 cell lines from Nuochen Biologicals Inc. in Shanghai.4.2.2 The expression changes of HDAC9 in the LSECs after OGD treatmentPlant the LSECs on the culture dish, divided the cells into two groups randomly, the control group and the OGD group. After reoxygenation, using Western Blot to detect the expression changes of HDAC9 after OGD treatment.4.3 The role of HDAC9 on LSECs dysfunction after OGD treatment4.3.1 After transfected the LSECs with shRNA-HDAC9, detect the transfection efficiency via Western Blot.4.3.2 After the successful transfection, detected the cells apoptosis via flow cytometric analysis under OGD treatment.Results:Part I:The expression change and fuction of HDAC9 in the cerebral I/RI1.1 The expression change of HDAC9 in the cerebral cortex after I/RI1.1.1 To build the rats MCAO modelThe Sprague-Dawley rats (250-280g) were randomly divided into two groups, the sham group and the MCAO group. The cerebral injury after surgery was confirmed by TTC staining and the neurological deficit score.1.1.2 The expression distribution of HDAC9 in the cerebral cortexThe expression distribution of HDAC9 was detected by immunofluorescence analysis. We identified neurons by staining for the neuronal nuclear marker NeuN, astrocyte marker GFAP, microglia marker CD11b and BMVECs maker CD34. We found that HDAC9 was expressed in all these cells with a relative low expression in microglia, which was further confirmed by RT-PCR and Western Blot.1.1.3 The expression changes of HDAC9 in the cerebral cortex after I/RIThe HDAC9 were detected by the Real time RT-PCR and Western Blot analysis after I/RI. We found that HDAC9 was markedly increased in the ischemic cerebral hemisphere.1.2 HDAC9 gene silencing via the intraventricular lentivirus injection using the stereotaxis instrument1.2.1 To build the HDAC9 gene silencing rat modelConstructed HDAC9 gene silencing vector, the shRNA-HDAC9 sequence was ATCATCCTGAGGTCTGTCC.1.2.2 To build the shRNA-HDAC9 rats models.We infused the pGLV3-shRNA-HDAC9 into the left cerebral cortex of rats by stereotaxicinjection. After 1 week injection, intense green fluorescence was seen in the cerebral cortex after delivery ofthe lentivirus pGLV3-shRNA-HDAC9 encoding GFP, but not in control brains rats which injected with PBS. In addition, we found the predominant localization of GFP expression in the left cortical areas.We further examined which kind of cells gene silencing occurred as demonstrated by GFP expression after stereotaxic injection of lentivirus through confocal immunofluorescent analysis using specific anti-GFP monoclonal rabbit antibody and antibodies for different celltype markers. Our results showed that GFP was expressed in different cerebral cells including neuron, microglia, astrocytes and BMVECs.The efficiency of HDAC9 gene silencing was further confirmed by Western Blot analysis. We found that HDAC9 levels were markedly reduced after transfection.1.3 The effect and potential mechanism of HDAC9 gene silencing on the ischemic stroke outcomes1.3.1 The effect of HDAC9 gene silencing on the ischemic stroke outcomesThe rats were divided into four groups randomly, induced ischemia in the left hemisphere by MCAO 1 week after the HDAC9 gene silencing. The rats were sacrificed 48 hour after the reperfusion. Using TTC and evans blue staining, we observed that HDAC9 increased the infarction volume, edema formation and BBB permeability dysfunction, as well as neurological deficits.1.3.2 Potential mechanism of HDAC9 gene silencing on the ischemic strokeFurthermore, using evans blue staining and the transmission electron microscopy analysis, we found that in ischemic rats, the endothelial cells and their nucleus were swollen and deformed, and the integrity of BBB was destroyed, presenting perivascular edema, vacuolation and membrane damage. However, gene silencing of HDAC9 ameliorated the endothelial injury and the BBB disruption after MCAO. Meanwhile, Western Blot indicated the increased expression of TJPs (ZO-1, Occludin and Claudin-5) was recovered after HDAC9 gene silencing.Part Ⅱ:The effect of HDAC9 on endothelial dysfunction under OGD condition2.1The Oxygen and Glucose Deprivation (OGD) model2.1.1 Primary Brain Microvessel Endothelial Cells (BMVECs) cultureWe used the rat primary BMVECs for study.2.2 The expression changes of HDAC9 in the BMVECs after the OGD injuryThe BMVECs were under OGD treatment, harvest the cells after different reoxygenation time, Western Blot shown that the HDAC9 was increased in a time-manner after OGD treatment.2.3 The role of HDAC9 gene silencing or overexpression on BMVECs injury after OGD treatmentWestern Blot showed the satisfied transfection efficiency after BMVECs transfected with shRNA-HDAC9 or pGV230-HDAC9. Using Real time RT-PCR, we found that OGD-induced pro-inflammatory mediators were attenuated by HDAC9 gene silencing. The flow cytometric analysis shown the BMVECs apoptosis was also attenuated by HDAC9 gene silencing.2.4 The role of HDAC9 on BMVECs permeability changes after OGD treatment2.4.1 Cell permeability assay indicated that gene silencing of HDAC9 ameliorated the OGD-induced abnormal endothelial cell permeability.2.4.2 Immunofluorescence analysis clarified the damaged ZO-1 protein was recovered after gene silencing of HDAC9, accompanied by increased expression of TJPs including ZO-1, Occludin and Claudin-5.Part Ⅲ:The functional role of HDAC9-mediated autophagy on BMVECs injury under OGD condition3.1 The regulation function of HDAC9 on BMVECs autophagyWestern Blot found that gene silencing of HDAC9 increased LC3Ⅱ/Ⅰ ratio and reduced p62 levels in BMVECs under OGD. We also examined p62 mRNA levels by Real time RT-PCR and found that gene silencing of HDAC9 had no effect on p62 mRNA expression, indicating that the decrease in p62 protein level is because of its degradation alongside polyubiquitinated proteins destined for autophagosomes rather than decreased transcription.Furthermore, Overexpression of HDAC9 decreased the LC3Ⅱ/Ⅰ ratio via Western Blot and the number of typical autophagosomes via transmission electron microscopy.3.2 The potential role of autophagy on BMVECs under OGD conditionThe mTOR inhibitor-rapamycin was added to activate the autophagy, using the flow cytometric analysis and Real time RT-PCR, we found that OGD significantly induced inflammatory response and endothelial cell permeability dysfunction, which were alleviated by rapamycin as well as by gene silencing of HDAC9.Part Ⅳ:The expression change and fuction of HDAC9 in liver after liver I/RI4.1 The expression change of HDAC9 in liver I/RI4.1.1 To build the rat liver I/R modelThe Sprague-Dawley rats (250-280g) were randomly divided into two groups, the sham group and the liver I/R group. After the operation, HE staining analysis found that in I/R group, the neutrophils were infiltrated, the liver cell membrane and nuclear were deformated, we also observe the significant hepatic apoptosis phenomenon.4.1.2 The expression change of HDAC9 after liver I/RITo examine the changes of HDAC9 in liver I/RI, Western Blot was proformed, our result shown that the HDAC9 was significantly increased after liver I/RI. Furthermore, the immunohistochemistry staining indicated that the HDAC9 was significantly increased after liver I/RI in the paffin section.4.2 The changes of HDAC9 on sinusoidal endothelial cells (LSECs) after OGD treatment4.2.1 To culture the LSECsWe use the RC-RM-0037 cell lines from Nuochen Biologicals Inc. in Shanghai for study.4.2.2 The expression change of HDAC9 in the LSECs after OGD injuryPlant the LSECs on the culture dish, divided the cells into two groups randomly, the control group and the OGD group. After reoxygenation, using Western Blot indicated that the HDAC9 was significantly increased after OGD treatment. Similar result was shown by the IHC staining.4.3 The role of HDAC9 on LSECs dysfunction after OGD treatment4.3.1 After transfected the LSECs with shRNA-HDAC9, detect the transfection efficiency via Western Blot.4.3.2 After the successful transfection, divided the cells randomly and stimulated individually. The flow cytometric analysis shown the ECs apoptosis was also attenuated by HDAC9 gene silencing.Conclusion:1. HDAC9 significantly increased in cerebral ischemic/reperfusion injury, HDAC9 increased cerebral injury in experimental stroke. HDAC9 is one of critical components of a signal transduction pathway that links cerebral injury to epigenetic modification in the brain.2. HDAC9 contributed to OGD induced BMVECs dysfunction as demonstrated by the increased inflammatory responses, cellular apoptosis and endothelial cell permeability dysfunction accompanied by reduced expression of tight-junction proteins.3. HDAC9 suppressed the endothelial protective autophagy, which was associated with endothelial dysfunction under OGD treatment.4. HDAC9 was also increased in the liver ischemic/reperfusion injury. HDAC9 contributed to the LSECs dysfunction under OGD condition, indicating that HDAD9 may have the same function in both ischemic diseases. |
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