Dexmedetomidine Improve Cognitive Function In Mice With POCD Via Regulating SPHK1

Objective:Postoperative cognitive dysfunction(POCD)is a common postsurgical complication.The incidence of POCD varies from 20-79% in cardiac surgery and 4.1-22.3% in non-cardiac surgery,according to data from epiderniologic studies.The occurrence of POCD is closely related to increased mortality,longer hospital stays,increased medical costs,and decreased quality of life.There is currently no particularly effective treatment for POCD.Dexmedetomidine(DEX)is a highly selective ?2 receptor agonist and is believed to have neuroprotective effects.A few of existing studies believe that DEX can treat POCD,but the mechanism is not yet clear.Metabolism is essential for growth,development and many key physiological functions.Metabolism has received increasing attention in recent years.The metabolic diseases existing before surgery are the risk factors of POCD.Metabolomics,as an emerging method in systems biology,is increasingly used in studying the underlying mechanisms of various diseases.There is no research on the effect of DEX on the metabolites of POCD mice.In this paper,we investigated the effect of DEX on cognitive function in mice with POCD and hippocampal metabolites in POCD mice.According to the results of metabolomics and database,an interaction network was constructed to search for key proteins and possible potential mechanisms.Finally,relevant verification was conducted through in vitro experiments.Methods:1.POCD model Construction and the effect of DEX on the behavioral performance of POCD mice:A POCD model was constructed by laparotomy on 12-month-old aged C57 BL / 6 mice.At the end of the operation,DEX was injected intraperitoneally,and behavioral procedures were performed by Morris water maze(MWM)and Open Field Test(OFT).Each mouse was given four swimming trials per day for five consecutive days after the laparotomy.Spatial acquisition was performed from the 1st day to the 5th day after the operation.OFT was performed on the 6th day after the operation,and probe trial was performed 30 minutes after the end of the OFT2.The effect of DEX on hippocampal metabolites in POCD mice:The effect of DEX on hippocampus of POCD mice was studied by metabolomics.Samples of fresh hippocampal tissue were obtained in different groups.Sample were applied to extraction procedures;supernatant was transferred to vial for LC-MS analysis.A LC-MS-based metabolomic approach was employed to determine the metabolic disorders of Control-Surgery and DEX+Surgery-Surgery..The data was performed feature extraction and preprocessed with Compound Discoverer software,and then normalized and edited into two-dimensional data matrix by excel 2010 software,The data after editing were performed Multivariate Analysis(MVA)using SIMCA-P software.The multivariate statistical analysis used in this study is mainly principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA),orthogonal partial least squares discriminant analysis(OPLS-DA),et.OPLS-DA was employed to establish a model for discriminating between control group,Surgery group and DEX+Surgery group.Metabo Analyst was used to identify the metabolic pathways that were associated with the identifed combinations of metabolites,thus indicating which pathways are important for the host response to DEX+Surgery group.Metabolites were input into the online database IPA,and an interaction network of metabolites were constructed according to the connections between the metabolites.3.The role of SPHK1 in the inhibition of inflammation by DEX:Firstly,BV2 cells were cultured in vitro.The dose of DEX on the expression of IL-6 and TNF-? were observed.The cells were stimulated with LPS and DEX for 6 hours.The supernatant was collected to detect the protein level of IL-6 and TNF-? by ELISA.Secondly,The effect of DEX on the expression of IL-6 and TNF-? m RNA and protein were observed.BV2 cells were cultured in vitro.The cells were stimulated with LPS and DEX for 1 hours to detect the m RNA level of IL-6 and TNF-? by QT-PCR.The cells were stimulated with LPS and DEX for 6 hours to detect the protein level of IL-6 and TNF-? by ELISA.Thirdly,The effect of DEX on the expression of SPHK1 m RNA and protein were observed.BV2 cells were cultured in vitro.The cells were stimulated with LPS and DEX for 1 hours to detect the m RNA level of SPHK1 by QT-PCR.The cells were stimulated with LPS and DEX for 6 hours to detect the protein level of SPHK1 by Western blotting.Finally,SPHK1 was activated by MHP to observe the effect of DEX on inflammatory cytokines.BV2 cells were cultured in vitro.The cells were stimulated with LPS,DEX and MHP for 6 hours to detect TNF-? and IL-6 by ELISA.Results:1.POCD model Construction and the effect of DEX on the cognitive functions of POCD mice:The results of the Spatial acquisition reflected that the mice in the Surgery group were slower and later familiar with the environment than the mice in the Control group.Results suggested that the POCD model was successfully constructed by laparotomy.The mice in the DEX + Surgery group can get familiar with the environment faster and earlier than the mice in the Surgery group.Results suggests that DEX improves the postoperative learning ability of POCD mice.The results of probe trails showed that the mice in the Surgery group had less accurate and less reliable memory of the platform than the mice in the Control group,suggesting that the POCD model was successfully constructed by laparotomy.The DEX + Surgery group mice had more accurate and firm memory of the platform than the Surgery group mice,suggesting that DEX improved the spatial memory ability of POCD mice.The results of the open field test suggest that there is no difference in the spontaneous behavior of the three groups of mice,and the effects of anxiety and depression on the results of the water maze experiment can be ruled out.2.The effect of DEX on hippocampal metabolites in POCD mice:12 samples of Surgery group,12 samples of DEX+Surgery group and 12 samples of Control group were included in the analysis of CO-metabolism.PCA analysis,PLS-DA analysis and OPLS-DA analysis showed that Control group,Surgery group and DEX+Surgery group showed a distinct trend of separation.18 different metabolites were screened at(ESI+)ion mode and 13 different metabolites were screened at(ESI-)ion mode in Surgery group vs Control group.18 different metabolites were screened at(ESI+)ion mode and 9 different metabolites were screened at(ESI-)ion mode in Surgery group vs DEX+Surgery group.Through the analysis of metabolites and metabolic pathways and the construction of an interaction network,we found that,as a rate-limiting enzyme for sphingolipid metabolism,SPHK1,is closely related to multiple differential metabolites and multiple signaling pathways such as MAPK and NF-?B.3.The role of SPHK1 in the inhibition of inflammation by DEX:In BV2,protein levels of TNF-? and IL-6 in the LPS group were significantly increased.TNF-? in the 10?M,50?M,100?M DEX treatment group were significantly lower than those in the LPS group.There was no significant change in the group of 0.1?M,1?M,5?M DEX compared with the LPS group.IL-6 in the1?M,5?M,10?M,50?M,100?M DEX treatment group were significantly lower than those in the LPS group.There was no significant change in the group of 0.1?M DEX compared with the LPS group.The results indicated that DEX inhibited the release of TNF-? and IL-6 induced by LPS at a certain dose.In BV2,the m RNA expression of TNF-? and IL-6 in the LPS group were significantly increased,but which in the 10 ?M DEX treatment group were significantly lower than those in the LPS group.In BV2,the m RNA expression and protein levels of SPHK1 in the LPS group were significantly increased,which were significantly lower in the 10?M DEX treatment group than those in the LPS group.In BV2,TNF-? and IL-6 in the 10 ?M DEX treatment group were significantly lower than those in the LPS group.When activating SPHK1,TNF-? and IL-6 in the LPS+DEX+MHP group were significantly higher than those in the10 ?M DEX treatmen group.Conclusion:Dexmedetomidine can improve the learning ability and spatial memory ability of POCD mice without affecting the spontaneous behavior.Dexmedetomidine treatment resulted in significant differences in metabolites in the hippocampus.In BV2 cells,dexmedetomidine exerts anti-inflammatory effects by inhibiting SPHK1,and activating SPHK1 can reverse the inhibitory effect of dexmedetomidine on inflammatory responses.