The Mechanism Of Caffeic Acid Phenethyl Ester In Alleviating Postoperative Cognitive Dysfunction Of Aged Mice

Background and objective: Postoperative cognitive dysfunction(POCD)is a serious neurological complication after anesthesia and surgery,mainly manifested as impairment of learning and memory function,mental disorder,anxiety,personality change,social dysfunction and so on.However,there is still a lack of effective clinical pharmacotherapy due to its unclear pathogenesis.Silent information regulator 6(Sirt6)is a highly conserved niacinamide adenine dinucleotide-dependent Class III histone deacetylase,which is related to brain aging,energy metabolism,and oxidative stress.In recent years,some studies have found that Sirt6 is involved in the occurrence and development of mood disorders and Parkinson’s disease.However,whether Sirt6 participates in the occurrence and development of POCD remains unclear.Additionally,since no commercial Sirt6-targeting drugs that can cross the blood-brain barrier exist,we sought to find a drug with the potential to play a similar role.Caffeic acid phenethyl ester(CAPE),which is a natural compound mainly extracted from propolis,plays a neuroprotective role to prevent the development of stroke and Alzheimer’s disease by reducing neuroinflammation and apoptosis.In this study,we aimed to explore whether Sirt6 is involved in the development of POCD,evaluate whether CAPE mitigated cognitive impairment following anesthesia and surgery through Sirt6/Nrf2 pathway,and explore its related mechanism.Methods:(1)Isoflurane anesthesia and tibial fracture surgery were used as the POCD model.Firstly,open field test(OFT)was performed to evaluate whether each group’s locomotor activity was consistent.Subsequently,Y-maze test(YMT)and Morris water maze test(MWMT)were used to access short-and long-term spatial learning and memory function,respectively.Additionally,reactive oxygen species(ROS)in the hippocampus and oxidative stress indicators,including catalase(CAT),malondialdehyde(MDA),glutathione(GSH),and superoxide dismutase(SOD),in the plasma,were evaluated by corresponding assay kits.Real-time quantitative polymerase chain reaction(RT-q PCR)and western blot(WB)were used to analyze the expressions of Sirt6 and Nrf2.Furthermore,immunofluorescence(IF)was used to explore Sirt6 localization and expression changes in neurons,microglia and astrocytes.(2)To investigate whether CAPE pretreatment could ameliorate cognitive dysfunction following anesthesia and surgery,CAPE was given(i.p.,10 mg/kg)before anesthesia and surgery for 10 consecutive days.OFT was performed to evaluate the locomotor activity of mice.Subsequently,YMT and MWMT were used to access spatial learning and memory functions.Besides,ROS in the hippocampus and oxidative stress indicators,including CAT,MDA,GSH,and SOD,in the plasma,were evaluated by corresponding assay kits.RT-q PCR and WB were used to analyze the expressions of Sirt6 and Nrf2 among the groups.IF was used to further explore the expression changes of Sirt6 in microglia and investigate the morphology and the number of microglia in the hippocampus.Furthermore,RT-q PCR was used to evaluate the expressions of M1 biomarkers,M2 biomarkers,proinflammatory cytokines and anti-inflammatory cytokines.(3)To further evaluate the crucial role of Sirt6 in the protective effect of CAPE,in vivo and in vitro studies were conducted.H2O2-induced BV2 cells were established as the microglial oxidative stress model.Specifically,the optimal concentration of hydrogen peroxide(H2O2)was selected by CCK-8 assays and FCM.Subsequently,to determine whether the microglial oxidative stress model could mimic changes in vivo,the changes of Sirt6 and Nrf2 expressions were detected by RT-q PCR and WB.CCK-8 and FCM were used to detect ROS levels,so as to judge the antioxidant ability of CAPE and screen out the optimal intervention concentration.Next,RT-q PCR,WB,and IF were used to access the effects of CAPE pretreatment on ROS levels,Sirt6/Nrf2 pathway and microglial polarization.Based on the results in vitro,in vivo experiments were carried out.OFT,YMT,and MWMT were performed to test whether Sirt6-specific inhibitor OSS＿128167 would reduce the neuroprotective effect of CAPE.Furthermore,RT-q PCR,WB,IF and other techniques were conducted to explore whether OSS＿128167 could regulate the level of oxidative stress indicators,Sirt6/Nrf2 pathway and microglial polarization.Results:(1)OFT results indicated no obvious difference in the total distance between the CON group and the A+S group.Furthermore,YMT results showed that anesthesia and surgery reduced the percentage of spontaneous alternation behavior in aged mice.MWMT results indicated that the escape latency increased and the number of platform crossings decreased in the A+S group,compared with the CON group.These results demonstrated that short-and long-term spatial memory of mice were impaired after anesthesia and surgery.The results from dihydroethidium(DHE)staining and corresponding biochemical assay kits indicated that anesthesia and surgery increased ROS generation in the hippocampus,increased MDA levels in the plasm,and decreased SOD,GSH and CAT levels in the plasm.Additionally,RT-q PCR and WB results showed that the expressions of Sirt6 and Nrf2 were decreased after anesthesia and surgery.Furthermore,IF results showed that the decreased expression of Sirt6 in hippocampus was mainly manifested in microglia and neurons.Besides,the decreased expression of Sirt6 in astrocytes only occurred in the hippocampal DG region.(2)OFT results indicated no obvious difference in the total distance among the CON group,A+S+Vehicle group and A+S+CAPE group.Furthermore,the results from YMT and MWMT showed that CAPE pretreatment ameliorated the decreased percentage of spontaneous alternation behavior,shortened the escape latency and enhanced the number of platform crossings.Thus,these results demonstrated that CAPE ameliorated short-and long-term cognitive impairment following anesthesia and surgery.Additionally,DHE staining results indicated that CAPE pretreatment notably eliminated ROS generation in the hippocampal CA1,CA3,and DG regions.Corresponding biochemical assay kits results indicated CAPE pretreatment dramatically increased the antioxidant(CAT and SOD)levels and reduced the MDA level in the plasm,compared with the A+S+Vehicle group.Besides,RT-q PCR and WB results showed that CAPE pretreatment increased the expressions of Sirt6 and Nrf2.IF results showed that the expressions of Sirt6 and Nrf2 in microglia were increased by CAPE pretreatment.Furthermore,IF results indicated that CAPE pretreatment decreased the number of microglia and facilitated the switch of hippocampal microglia from the M1 to the M2 type.(3)H2O2-induced BV2 cells were successfully established as the microglial oxidative stress model for subsequent experiments.FCM results demonstrated that CAPE pretreatment notably eliminated ROS generation in H2O2-induced BV2 cells,which would be attenuated by a specific Sirt6 inhibitor,OSS＿128167.Additionally,RTq PCR,IF,WB and FCM results indicated that CAPE pretreatment promoted the switch of H2O2-induced BV2 cells from the M1 to the M2 type through activating Sirt6.Furthermore,in vivo experiments were carried out.OFT,YMT and MWMT results indicated that OSS＿128167 reduced the neuroprotective effect of CAPE,including short-and long-term spatial memory.Additionally,RT-q PCR,WB,IF and other techniques demonstrated that OSS＿128167 leaded to oxidative stress response and microglial M1 polarization and inhibited the Sirt6/Nrf2 pathway.Conclusions:(1)The experiments demonstrated that anesthesia and surgery impaired shortand long-term spatial memory.Besides,anesthesia and surgery increased oxidative stress response in the hippocampus and in the plasm.Anesthesia and surgery inhibited the hippocampal Sirt6/Nrf2 pathway,especially in microglia and neurons.(2)The experiments indicated that CAPE pretreatment suppressed oxidative stress and promoted M2 microglia polarization via Sirt6/Nrf2 pathway to mitigate cognitive impairment in aged mice following anesthesia and surgery.Moreover,further mechanistic studies in BV2 cells and aged mice indicated that OSS＿128167 reduced the neuroprotective effect of CAPE on reducing ROS generation and promoting protective polarization.Besides,OSS＿128167 reversed short-and long-term spatial memory alleviated by CAPE following anesthesia and surgery.