Up-regulation Of Prdx6 By Cerebral Ischemia-reperfusion And Its Correlation With Inflammatory Factors

Peroxiredoxin-6,a member of the peroxidase reductase family,is a protein with dual enzyme activity that has both phospholipase A2 and peroxidase reductase activity.Several articles have demonstrated that Prdx6 plays an important role in various diseases such as Parkinson's disease,Jakob disease,Pick's disease and malignant mesothelioma of lung.However,the role of Prdx6 in stroke has not been well studied.In this study,the cerebral ischemic model was proved by using 2,3,5-triphenyltetrazolium chloride staining(TTC)and hematoxylin-eosin staining(HE).Immunohistochemistry(IHC)was used to observe the expression of Prdx6 in different regions of the brain.Western blot(WB)and real-time quantitative PCR(q RT-PCR)were used to detect the protein and m RNA levels of Prdx6 respectively in the different regions of the brain.Immunofluorescence double labeling was used to observe the expression of Prdx6 in neurons,microglia,and astrocytes.The expressions of i NOS,TNF-?,IL-1,IL-6 and IL-10 m RNA were detected by RT-PCR,and the correlation with Prdx6 expression in different brain regions of rats with middle cerebral artery occlusion(MCAO)were analyzed.Objective: 1.To observe Prdx6 expression in different brain regions;2.To observe the cell types that expresses Prdx6 in different brain regions;3.To investigate whether there is a correlation between Prdx6 expression and inflammatory factors in the brain of MCAO rats.Methods: A model of transient focal cerebral ischemia and reperfusion was established in rat by MCAO with an intravascular suture method.HE staining was used to detect ischemic and necrosis tissue using TTC staining and HE staining.Immunohistochemistry was used to observe the expression of Prdx6 in different brain regions.Western blot and real-time fluorescence quantitative PCR were used to detect Prdx6 protein and m RNA levels,respectively.Immunofluorescence double labeling method was used to observe the expression and localization of Prdx6 in neurons,microglia and astrocytes.q RT-PCR was used to detect the m RNA levels of inflammatory cytokines in brain regions of MCAO mice and to analyze their correlation with Prdx6 expression.Results: 1.The MCAO model was successfully established To determine whether the rat MCAO model is successfully established,we firstly performed TTC staining and HE staining.TTC staining revealed that the first,second,third and fourth slices of the brain slices of MCAO rats had necrotic areas and the fourth necrotic area reached the maximum.HE staining was used to detect the brain specific necrotic area in the MCAO rats and found that it was focused on the first somatic sensory cortex and the second somatic sensory cortex.2.Astrocytes and microglial cells were activated in MCAO brain and neurons were apparently apoptotic Using the same batch of paraffin sections as those observed by HE staining,we first examined whether the astrocytes in the cerebral cortex,hippocampus,thalamus and hypothalamus of MCAO rats were activated by immunohistochemistry.It was shown that the astrocytes in the cerebral cortex,hippocampus,thalamus and hypothalamus were significantly activated in the MCAO group compared with the sham group.Then,IHC analysis showed that the microglia of the cerebral cortex,hippocampus and hypothalamus were activated in the MCAO group,and microglia in the hypothalamus are significantly activated.Finally,the morphology of neurons in cerebral cortex of MCAO rats were detected by IHC analysis,immunohistochemistry results showed that compared with sham rats neuronal cells in the cerebral cortex of MCAO rats were significantly apoptosed.The above experimental results are statistically significant.3.Up-regulation of Prdx6 expression in MCAO brain Immunohistochemistry showed that Prdx6 expression was up-regulated in rat brains at 2 hours of ischemia compared with the sham controls.Compared with reperfusion 0 h and 6 h,the expression of prdx6 was significantly up-regulated in brains at 12 h and 24 h(P < 0.05).The expression of Prdx6 was significantly up-regulated at 12 h and 24 h after reperfusion,compared with that at 0 h and 6 h by using WB.4.Expression of Prdx6 in different brain regions in MCAO rats To investigate the expression of Prdx6 in different brain regions of the MCAO model,we examined Prdx6 expression in the cerebral cortex,hippocampus,thalamus,and hypothalamus.We found that Prdx6 expression in the cortex and hippocampus in the MCAO group was higher than that in the sham group.Prdx6 has a stronger immunofluorescence signal in the thalams than that in other brain regions.5.Prdx6 was mainly expressed in astrocytes in MCAO model rats We used double-labeling immunofluorecent staining to observe the cell types that express Prdx6.Neuronal-specific nuclear protein(Neu N),lonized calcium-binding adapter molecule 1(iba-1),and glial fibrillary acidic protein(GFAP)were used as the markers for neuron,microglia,and astrocyte,respectively.We found that Prdx6 was mainly expressed in astrocytes both in the normal and MCAO rats.It was rarely expressed in the neurons and microglial cells.6.Prdx6 expression is related with inflammatory factors in MCAO model rats We first detected m RNA levels of Prdx6 and various inflammatory cytokines(i NOS,TNF-?,IL-1,IL-6,IL-10)by RT-PCR.We found the expression of prdx6 m RNA in the hypothalamus was up-regulated.The expression of i NOS m RNA was increased in the cortex and thalamus,but decreased in hypothalamus,and was not significant changed in hippocampus.TNF-? was increased in hippocampus and thalamus,not significantly changed in other regions.IL-1 was increased in cortical,but decreased in thalamus and hypothalamus,and not significantly changed in hippocampus.IL-6 was increased in hippocampus and thalamus,and was not significantly changed in other brain regions,IL-10 was increased in the thalamus,but decreased in hypothalamus,and not significantly changed in other brain regions.We then performed a correlation analysis of these inflammatory factors and Prdx6 and found that Prdx6 is positively correlated with i NOS and IL-1.It is negatively associated with TNF-?,IL-6,and IL-10.Conclusions: 1.Prdx6 was up-regulated in the brains of rats after cerebral ischemia-reperfusion,and it mainly located in cortex,hippocampus,thalamus and hypothalamus.2.Prdx6 is mainly expressed in astrocytes and rarely expressed in small amounts in piriform cortical neurons.3.There is a positive correlation between Prdx6 and inflammation factors(i NOS,IL-1),and Prdx6 is negatively related to inflammatory factors such as TNF-?,IL-6 and IL-10 in brain of MCAO rats.