The Effect Of Prdx2 And Its Mechanism On Sepsis-associated Encephalopathy

Chapter 1 Prdx2 expression in the hippocampus of mice following septic encephalopathyObjective: We performed Cecal Ligation and Puncture(CLP)experiment to establish Sepsis-Associated Encephalopathy(SAE)model in mice.The expression of Peroxiredoxin-2(Prdx2)in the hippocampus of SAE mice was determined at different time points.Methods: The SAE model was established in SPF C57BL/6 male mice.The mice were performed with sham and CLP procedure.Behavioral tests were conducted 7 days after surgery.Open field test(OFT)was applied to assess the level of autonomous activity.Morris water maze(MWM)and Fear conditioning test(FCT)were used to assess learning and memory function.Another group of SPF C57BL/6 male mice were randomly divided into sham group,6h,12 h,1d,3d,5d and 7d CLP mouse model groups,and the mice were sacrificed at6 h,12h,1d,3d,5d and 7d,respectively.Western Blot and real-time PCR were used to observe the expression of Prdx2 in SAE mouse brain tissues,and immunofluorescence staining was used to determine the distribution cells of Prdx2 in brain tissues.Results:(1)In the open field test,sepsis did not affect the autonomous motor ability of mice.In the Morris water maze,compared with sham group,CLP group showed longer escape latency,spent less time in target quadrant and had fewer numbers of crossing the platform.As with the fear conditioning test,CLP group exhibited shorter freezing time than sham group.(2)Western Blot results indicated that Prdx2 expression was significantly decreased at 12h-7d after CLP,most obviously at 24 h after CLP.Real-time PCR results demonstrated a significant decrease in Pdx2 gene expression at 6h,12 h,1d,3d after CLP.(3)The double immunofluorescence stain showed that Prdx2 mainly colocalized with hippocampal neurons and only a small amount of Prdx2 was observed in astrocytes and microglia.The expression of Prdx2 in hippocampal neurons in CLP group was significantly lower than that in sham group.Conclusion:(1)CLP mouse model demonstrated significant cognitive impairment,implying that SAE model was successfully established.(2)Prdx2 was mainly expressed in neurons of brain tissues,and CLP mice showed a dramatic down-regulation of Prdx2.Chapter 2 The effect of Prdx2 on SAE-induced brain damage in miceObjective: To investigate the effect of Prdx2 on sepsis induced brain injury in mice.Methods: The SAE model was established in SPF C57BL/6 male mice.They were randomly divided into sham group(mice were injected into hippocampus with normal saline at 0.6ul per each side.3 weeks later mice received sham operation),CLP group(mice were injected into hippocampus with normal saline at 0.6ul per each side.3 weeks later mice received CLP operation),CLP+Prdx2 group(mice were injected into hippocampus with AAV-Prdx2 at 0.6ul per each side.3 weeks later mice received CLP operation),CLP+sh Prdx2 group(mice were injected with AAV-sh Prdx2 at 0.6ul per each side.3 weeks later mice received CLP operation).24 h after surgery,hippocampal tissues of each group were collected for western blot and immunofluorescence staining to confirm the expression of Prdx2 in mouse brain tissues.The apoptosis related proteins(cleaved caspase-3,Bax,Bcl2),autophagy related proteins(LC3B,p62,Beclin-1)in brain tissues were determined by western blot.Nissl staining was applied to observe the effect of Prdx2 overexpression or knockdown on hippocampal neuron.Autophagy of hippocampal neurons was detected by transmission electron microscopy.Brain inflammation and oxidative stress levels(TNF-α,IL-1β,IL-6,SOD,MDA)were analyzed by Elisa.Behavioral tests were performed 7 days after surgery.Results:(1)Western Blot and immunofluorescence staining results showed that AAV2-Prdx2 increased Prdx2 expression in the brain tissues of SAE mice,whereas AAV2-sh Prdx2 dramatically inhibited it 24 hours after CLP surgery.(2)Compared with the CLP group,Prdx2 overexpression significantly decreased apoptosis of hippocampal neurons,reduced the expression of cleaved caspase-3,Bax and the levels of IL-1β,IL-6,MDA,promoted Bcl2 expression and SOD levels.(3)Compared with CLP group,Prdx2 knock-down dramatically enhanced apoptosis of hippocampal neurons,increased the expression of cleaved caspase-3,Bax and the levels of IL-1β,IL-6,MDA,decreased Bcl2 expression and SOD levels.(4)Compared with sham group,CLP group showed an increased number of autophagy vacuole,enhanced the expression of LC3II/LC3 I and Beclin-1,and inhibited the expression of p62,suggesting the activation of autophagy in hippocampus.Prdx2 overexpression further promoted autophagy,while Prdx2 knockdown reduced autophagy.(5)In the open field test,sepsis and overexpression or knockdown of Prdx2 did not affect the autonomous motor ability of mice.In the fear conditioning test,CLP group exhibited shorter freezing time than sham group.Mice in the CLP+Prdx2 group showed remarkably longer freezing time than CLP group while mice in the CLP+sh Prdx2 group exhibited less freezing time than CLP group.In the Morris water maze,compared with sham group,CLP group showed longer escape latency,spent less time in target quadrant and had fewer numbers of crossing the platform.Compared with CLP group,mice in the CLP+Prdx2 group required less time to find the platform while mice in the CLP+sh Prdx2 group required more time.AAV2-Prdx2 injected mice remained longer in target quadrant and crossed the platform more frequently than CLP mice,whereas the mice treated with AAV2-sh Prdx2 showed the opposite results.Conclusion: Prdx2 promotes neuron autophagy,inhibits neuroinflammation,oxidative stress and neuronal apoptosis,thus alleviates sepsis-induced cognitive impairment.Chapter 3 The mechanism underlying Prdx2 alleviates SAE-induced brain damage in miceObjective: To explore the underlying mechanism of Prdx2 in alleviating sepsis induced brain injury,and to determine the role of ASK1/p38/m TOR signaling pathway.Methods: The experiment was divided into three parts.(1)To investigate the influence of Prdx2 on ASK1/p38/m TOR signaling pathway in the hippocampus of septic mice.We chose SPF C57BL/6male mice to establish SAE model induced by CLP.They were randomly divided into sham group,CLP group,CLP+Prdx2 group,CLP+sh Prdx2 group.24 hours after surgery,hippocampal tissues of each group were collected for western blot and immunofluorescence staining to detect the expression of p-ASK1、p-p38、p-m TOR.(2)To observe the effect of p38/m TOR activation on Prdx2-induced hippocampal autophagy and Prdx2-mediated attenuation of SAE-induced brain damage.We chose SPF C57BL/6 male mice to establish CLP-induced SAE model.They were randomly divided into CLP group(mice were injected into hippocampus with normal saline at 0.6ul per each side.CLP operation was performed at 3 weeks later,then normal saline was injected at 1ul in hippocampus after surgery)and CLP+Prdx2 group(mice were injected into hippocampus with AAV-Prdx2 at 0.6ul per each side.CLP operation was performed at 3 weeks later.Then mice were injected with 1ul normal saline in hippocampus after surgery),CLP+Prdx2+P79350 group(mice were injected into hippocampus with AAV-Prdx2 at 0.6ul per each side.CLP operation was performed at 3 weeks later,and p38 agonist P79350 was injected into hippocampus with the total volume of 1ul after surgery),CLP+Prdx2+3DBO group(mice were injected with AAV-Prdx2 in hippocampus at 0.6ul per each side.CLP operation was performed at 3weeks later,and m TOR agonist 3DBO was injected in hippocampus with the total volume of 1ul after surgery).At 24 h after surgery,the hippocampal tissues of each groups were collected and determined by Western Blot,Nissl staining and Elisa.Behavioral tests were performed during 7 to 14 days postoperatively.(3)To observe the effect of p38/m TOR inactivation on sh Prdx2-induced downregulation of hippocampal autophagy and sh Prdx2-mediated aggravation of SAE-induced brain damage.We selected SPF C57BL/6 male mice to establish CLP-induced septic encephalopathy model.They were randomly divided into CLP group(mice were injected into hippocampus with normal saline at 0.6ul per each side.CLP operation was performed at 3 weeks later,and normal saline was injected at 1ul in hippocampus after surgery)and CLP+sh Prdx2 group(mice were injected into hippocampus with AAV-sh Prdx2 at 0.6ul per each side.CLP operation was performed at 3 weeks later.Mice were injected into hippocampus with 1ul normal saline after surgery),CLP+sh Prdx2+SB203580 group(mice were injected with AAV-sh Prdx2 at 0.6ul per each side.CLP operation was performed at 3 weeks later,and p38 inhibitor SB203580 was injected in hippocampus with the total volume of 1ul after surgery),CLP+sh Prdx2+Rapamycin group(mice were injected with AAV-sh Prdx2 via hippocampus at 0.6ul per each side.CLP operation was performed at3 weeks later,and m TOR inhibitor Rapamycin was injected in hippocampus with the total volume of 1ul after surgery).24 h after surgery,the hippocampal tissues of each groups were collected and determined by Western Blot,Nissl staining and Elisa.Behavioral tests were performed 7days after surgery.Results:(1)Results from Western Blot and immunofluorescence staining showed that compared with CLP group,Prdx2 overexpression decreased the expression of p-ASK1、p-p38、p-m TOR in hippocampus,while Prdx2knock-down enhanced the expression of p-ASK1、p-p38、p-m TOR in hippocampus.(2)Compared with CLP+Prdx2 group,the expression of p-p38 was increased in CLP+Prdx2+P79350 group,but there was no significant difference in p-ASK expression.The expression of p-m TOR increased in CLP+Prdx2+3DBO group,but there was no significant difference in the expression of p-p38 and p-ASK.P79350 and 3DBO significantly blocked Prdx2-mediated up-regulation of LC3II/LC3 I,Beclin-1,and down-regulation of p62 expression.Furthermore,P79350 and 3DBO both enhanced the expression of cleaved caspase-3 and Bax,reduced Bcl2 expression,promoted hippocampal neuron injury and apoptosis.Elisa results demonstrated that IL-1β 、 TNF-α 、 IL-6 、 MDA levels were significantly increased and SOD levels declined in P79350 and3DBO-injected hippocampi of mice compared to mice with Prdx2 overexpression.(3)Compared with CLP+sh Prdx2 group,the expression of p-p38 in CLP+sh Prdx2+SB203580 group was decreased,but the expression of p-ASK showed no significant difference.The expression of p-m TOR was decreased in CLP+sh Prdx2+Rapamycin group,while the expression of p-p38 and p-ASK showed no significant difference.SB203580 and Rapamycin rescued sh Prdx2-mediated down-regulation expression of LC3II/LC3 I,Beclin-1 and up-regulation expression of p62.SB203580 and Rapamycin reduced the expression of cleaved caspase-3 and Bax,enhanced Bcl2 expression,attenuated hippocampal neuron damage and apoptosis.A decrease in the levels of IL-1β、TNF-α、IL-6、MDA and an increase in the SOD level were detected in SB203580 and Rapamycin injected hippocampi of mice compared to mice with Prdx2 knockdown.(4)Behavioral tests showed that P79350 or 3DBO prevented Prdx2 overexpression mediated cognitive protection from SAE.However,SB203580 or Rapamycin treatment rescued Prdx2 knockdown mediated exacerbation of cognitive impairment.Conclusion: Prdx2 promotes autophagy of hippocampal neurons through ASK1/p38/m TOR signaling pathway,and inhibits neuroinflammation,oxidative stress and neuronal apoptosis,thus alleviating cognitive impairment in SAE mice.