The Mechanism Of Sevoflurane Induces Cognitive Dysfunction By Modulating Microglial Polarization In Aged Micearthritis

BackgroundPerioperative neurocognitive disorders(PNDs),which manifest as declines in perioperative attention,memory,executive ability and other cognitive functions,are a severe complication in the perioperative period of elderly patients.PNDs delay the postoperative recovery process,deteriorate life quality and even increase the mortality of patients.Unfortunately,there remains no ideal method to prevent and treat PNDs.Therefore,it is necessary to clarify the underlying mechanism of PNDs.Recently,accumulating evidence has revealed that neuroinflammation induced by anesthesia and surgery is involved in PNDs.Microglia,the innate immune cells in the central nervous system,survey the brain microenvironment and play an important role in maintaining homeostasis.When foreign pathogen insult occurs,normal "resting" microglia are rapidly activated and transformed into proinflammatory M1 and anti-inflammatory M2 phenotypes.Sevoflurane is one of the commonly used inhalational general anesthetics in surgery.Recently,multiple studies have shown that sevoflurane promote the secretion of various inflammatory factors and reactive oxygen species(ROS)by stimulating microglial Ml polarization,which leads to hippocampal neurons apoptosis and PNDs.However,the potential molecular mechanisms remain unclear.MicroRNAs(miRNAs)are small endogenous non-coding RNAs,which regulate the translation and expression process of downstream target genes by binding to the 3’-UTR of mRNA.MiRNAs participate in various biological processes,including cell proliferation,differentiation,apoptosis and tissue development.Notably,miR-125b-5p,one of the new members of miRNAs,is abundant in microglia and closely related to microglial M1 polarization.However,there is still some controversy about the role of miR-125b-5p in regulating microglial inflammation and cognitive dysfunction,and more further studies are required.This study will further elucidate the potential role of miR-125b-5p in sevoflurane-induced microglial neuroinflammation and cognitive impairments both in vivo and in vitro,which provide a theoretical basis for new strategies of PNDs.PurposeWe aimed to explore the role of miR-125b-5p in sevoflurane-induced microglial neuroinflammation and neurocognitive disorders,clarify the underlying mechanism of sevoflurane-mediated microglial M1/M2 polarization,and investigate the relationship between microglial polarization and neuronal damage.Methods1.Establishment of the aged mice model with different sevoflurane inhalation durations.Mice were exposed to 3%sevoflurane for 2 h,4 h and 6 h.To evaluate the microglial M1/M2 polarization in the hippocampus,western blotting was conducted to detect the protein expression levels of Iba1,CD86 and CD206,qRT-PCR was performed to measure the mRNA levels of IL-6,IL-1β,TNF-α,IL-10,Arg-1 and TGF-1β.The level of miR-125b-5p in the hippocampus was detected by qRT-PCR.The Morris water maze(MWM)and open field test(OFT)were performed to evaluate the cognitive function of mice in control and 6 h groups.2.Establishment of the sevoflurane-exposed aged mice model with low expression of miR125b-5p.Mice were anesthetized and injected intracerebroventricularly with miR-125b-5p antagomiR interference lentivirus vector.when the level of miR-125b-5p was downregulated,they were exposed to 3%sevoflurane for 6 h.3.Evaluation of the microglial M1/M2 polarization,hippocampal neuron damage and cognitive function.To evaluate the microglial M1/M2 polarization in hippocampus,immunofluorescence staining was used to observe the number of microglia,Western blotting was performed to detect the protein expression levels of Ibal,CD86 and CD206,qRT-PCR was conducted to measure the mRNA levels of IL-6,IL-1β,TNF-α,IL-10,Arg-1 and TGF-1β.To assess neuronal damage caused by microglial polarization in the hippocampus,the ROS level was detected using ROS/RNS kits,the protein expression levels of BDNF,Cleaved caspase-3,Bcl-2,Bax and SYP were measured by western blotting,and the apoptotic cells were observed by TUNEL staining.The MWM and OFT were performed to evaluate the cognitive function.4.Role of A20/NF-κB pathway in sevoflurane-induced microglial M1/M2 polarization.The downstream target gene A20 of miR-125b-5p were predicted through the TargetScan website and confirmed using Dual-luciferase Reporter Gene Assay.To clarify the role of A20/NF-κB pathway in sevoflurane-induced microglial M1/M2 polarization,the protein levels of A20,p-p65 and p65 in hippocampus were measured by Western blotting.5.Establishment of the inhaled sevoflurane model in BV2 cell lines with low expression of miR-125b-5p or low expression of both miR-125b-5p and A20.To further explore the roles of miR-125b-5p and A20/NF-κB pathway in sevofluraneinduced microglial M1/M2 polarization,BV2 cells were performed to an in vitro experiment.MiR-125b-5p inhibitor and miR-125b-5p inhibitor plus small interfering RNA against A20(siA20)were transfected into BV2 cells.Twelve hours after transfection,the cells were transferred to a sealed box in a 37℃ incubator and exposed to sevoflurane with 95%O2 and 5%CO2.Finally,BV2 cells were collected 24 h later for further analysis.To clarify the M1/M2 polarization of BV2 cells,the morphology of BV2 cells was observed under microscope,immunofluorescence staining and flow cytometry were performed to detect the fluorescence intensity of CD86 and CD206.The protein levels of A20,p-p65 and p65 were determined by Western blotting.6.Exploration of the relationship between microglial polarization and neuronal damage by Transwell Assay.A Transwell coculture system was conducted to verify the neurotoxicity of polarized BV2 cells.BV2 cells were transfected with miR-125b-5p inhibitor and subjected to sevoflurane exposure,then they were co-cultured with primary hippocampal neurons for 48 hours.ELISA was applied to measure the protein concentrations of inflammatory cytokines and BDNF in neuronal medium.RNS/ROS kit was preformed to determine the level of ROS in neuronal medium.The protein expression levels of Cleaved caspase-3、Bcl-2、Bax and SYP were detected by Western blotting.The apoptotic cells were detected by flow cytometry.Results1.Prolonged sevoflurane exposure upregulated the level miR-125b-5p,promoted M1 polarization and inhibited M2 polarization in microglia in the hippocampus,which led to neuroinflammation and cognitive dysfunction in aged mice.Sevoflurane increased the protein levels of Ibal and CD86,and decreased the protein levels of CD206 in the hippocampus.After sevoflurane exposure,the levels of proinflammatory cytokines IL-6,IL-1β and TNF-α were upregulated and the levels of anti-inflammatory cytokines IL-10,Arg-1 and TGF-1β were downregulated.The MWM showed that sevoflurane prolonged the escape latency in the place navigation,reduced the number of times crossing the platform and dwelling time in the target quadrant in the spatial probe test.The OFT revealed that sevoflurane reduced the residence time and distance of mice in the central area.2.Inhibition of miR-125b-5p ameliorated sevoflurane-induced neuroinflammation and promoted BDNF secretion by altering microglial M1/M2 polarization via the A20/NF-κB axis.Dual-luciferase Reporter Gene Assay confirmed that A20 was the downstream gene of miR-125b-5p.Compared with the mice exposed to sevoflurane,inhibition of miR-125b-5p reduced the number of microglia in hippocampus,decreased the protein expression levels of Ibal,CD86,p-p65 and p65,increased the levels of CD206,A20 and BDNF,downregulated the mRNA levels of IL-6,IL-1β and TNF-α and upregulated the levels of IL-10,Arg-1 and TGF1β.3.Inhibition of miR-125b-5p attenuated sevoflurane-induced neuronal damage in the hippocampus and improved cognitive function in aged mice.Compared with the mice exposed to sevoflurane,inhibition of miR-125b-5p decreased the number of apoptotic cells in the hippocampus,reduced the protein levels of Cleaved caspase-3 and Bax,elevated the protein levels of Bcl-2 and SYP,shortened the escape latency in the place navigation,increased the number of times crossing the platform and dwelling time in the target quadrant in the spatial probe test,and increased the total movement distance and residence time in the central area in OFT.4.Inhibition of miR-125b-5p alleviated sevoflurane-induced M1/M2 polarization in BV2 cells through the A20/NF-κB pathway.After sevoflurane exposure,more amoeboid BV2 cells were observed under microscopy,the fluorescence intensity of CD86 was enhanced and the intensity of CD206 was weakened,the protein level of A20 was reduced while the levels of p-p65 and p65 were increased.Inhibition of miR-125b-5p alleviated sevoflurane-induced M1/M2 polarization in BV2 cells,whereas transfection with si-A20 abolished the protective effect of miR-125b-5p inhibitor.5.Inhibition of miR-125b-5p attenuated sevoflurane-induced microglial neuroinflammation and promoted BDNF secretion,which alleviated neuronal damage.Compared with the sevoflurane-exposed BV2 cells,inhibition of miR-125b-5p downregulated the levels of IL-6,IL-1β,TNF-α and ROS,upregulated the levels of IL-10,Arg1 and TGF-1β and promoted BDNF secretion,which reduced the number of apoptotic cells,decreased the protein levels of Cleaved caspase-3 and Bax,and increased the levels of Bcl-2 and SYP in the co-cultured neurons.Conclusion1.Sevoflurane induces neuroinflammation and cognitive dysfunction in aged mice by altering microglial M1/M2 polarization.2.Sevoflurane modulates microglial M1/M2 polarization by upregulating the level of miR-125b-5p and activating the A20/NF-κB pathway.3.Inhibition of miR-125b-5p attenuates sevoflurane-induced microglial inflammation and promotes BDNF secretion,which alleviates neuronal damage and improves cognitive function.