The Research On The Role Of Phosphatidylserine In Perioperative Neurocognitive Disorders

BackgroundThe clinical manifestations of patients,especially elderly patients,which are characterized by perioperative lack of concentration,decreased language comprehension,cognitive decline and difficulty in returning to preoperative animation were named postoperative cognitive dysfunction(POCD)in 1998,and renamed perioperative neurocognitive disorders(PND)in 2018.Although the content of this study is neurocognitive dysfunction occurring in a short period of time after operation,which accords with the concept of POCD,some scholars suggest that should be named postoperative neurocognitive disorders.On the basis of the consensus of experts,in order to standardize and facilitate the narrative,we use the name PND except for the first chapter,in which the experiment was carried out earlier and at that time there was no PND concept yet.The pathogenesis of PND is currently unclear,possible mechanisms include inflammation of the central nervous system,oxidative stress,graceful degradation of the central cholinergic system,accumulation of amyloid β-protein and excessive phosphorylation of tau protein.It is currently believed that surgery affects the secretion of hypothalamic-pituitary-adrenal axis hormone,making carbohydrate,protein,fat,salt and water balance disorder,which may be a potential cause of PND.In this study,to analyze the serum of elderly patients with early POCD by using Ultra Performance Liquid Chromatography and Q-TOF Mass Spectrometry to identify the differentially expressed metabolites undergoing laparoscopic surgery for Gastrointestinal malignancies under general anesthesia.Identify the serological markers of early POCD and explore the pathogenesis of POCD.According to the discovery of potential markers,targeted intervention measures were taken,observe whether the intervention can improve PND and explore the mechanism through the elderly rat model undergoing splenectomy,in order to provide reference for clinical prevention and improvement of PND.Part Ⅰ Serum metabolomics of early postoperative cognitivedysfunction in elderly patientsObjective:To analyze the serum of patients with early postoperative cognitive dysfunction by using Ultra Performance Liquid Chromatography and Q-TOF Mass Spectrometry to identify the differentially expressed metabolites undergoing laparoscopic surgery for Gastrointestinal malignancies under general anesthesia.Identify the serological markers of early POCD and explore the pathogenesis of POCD.Materials and methods:40 patients scheduled to undergo laparoscopic surgery for gastrointestinal malignancies,aged over 65 years,and the preoperative score of the mini-mental state examination scale(MMSE)was not less than 23.All patients under general anesthesia by endotracheal intubation and perioperative management followed the requirement of enhanced recovery after surgery(ERAS).MMSE and Montreal Cognitive Assessment(MoCA)were performed 1 day before and 7 days after operation.Patients were divided into two groups according to the MoCA score.POCD group:MoCA score not over 24;Non-POCD group:MoCA score over 24.Finally,20 patients undergoing laparoscopic surgery for gastrointestinal malignancies were judged to be POCD and the incidence of POCD was 50%.Blood samples were obtained on the 7th postoperative day while Montreal Cognitive Assessment was performed,Liquid Chromatography(Agilent 1290)was equipped with Q-TOF/MS(Agilent 6538),which was developed for the determination of serum metabolites.The variables with VIP>1(variable importance in the projection)were potential differential metabolites.Furthermore,a t-test was used to determine if the differential metabolites selected from PLS-DA modeling were statistically significant(p<0.05).To identify potential markers,the following database was used:HMDB(http://www.hmdb.ca/)and METLIN(https://metlin.scripps.edu/index.php).Differential metabolite pathway analysis and interaction network analysis were completed with IPA software(IPA,Ingenuity(?)Systems,https://www.ingenuity.com).MetaboAnalyst 3.0(https://www.metaboanalyst.ca/)was used to assess the specificity and sensitivity of potential biomarkers with receptor operating characteristic(ROC)analysis.Results:40 differentially expressed metabolites were identified in the serum of the POCD group compared to the non-POCD group.These metabolites were mainly fatty acids,lipids,carnitine derivatives.ROC curve analysis indicated that phosphatidylserine(17:2/0:0),with an AUC value of 0.966,was considered a potential diagnostic marker for POCD.The interaction network of differentially expressed metabolites between the POCD and non-POCD groups was built based on the database of IPA.The five top canonical pathways included the nitric oxide signaling pathway,the PI3K-AKT signaling pathway,the mTOR signaling pathway,mitochondrial dysfunction and nuclear transcription factor-kappa B(NF-κB)signaling pathway.Conclusion:A serum metabonomics method based on UPLC-Q-TOF-MS was established to study the metabolic changes in POCD patients.The metabolic pathways perturbed in POCD have been reported.Forty differential metabolites were identified,fatty acids,lipids,carnitine derivatives significantly changed in POCD patients and PS(17:2/0:0)with an AUC value of 0.966 was the potential biomarker for POCD diagnosis.The interaction network analysis of these differently expressed metabolites in POCD was established by IPA.The results suggested that activations of the nitric oxide signaling pathway,the PI3K-AKT signaling pathway,the mTOR signaling pathway,mitochondrial dysfunction and NF-κB signaling pathway were responsible for the pathogenesis of POCD.Part Ⅱ The effect of phosphatidylserine intervention to perioperative neurocognitive disorders and oxidative stress in the hippocampus in elderly rats undergoing splenectomyObjective:To investigate whether phosphatidylserine intervention may improve perioperative neurocognitive disorders by inhibiting oxidative stress in the hippocampus of elderly rats undergoing splenectomy.Materials and methods:80 male aged Sprague-Dawley(SD)rats(CL),weight:380-420g,age:18 months and above.Rats were divided into four groups according to the random principle:control group(group C,n=20,conventional feeding for 30 days);anesthesia group(group A,n=20,resuscitation after intraperitoneal propofol injection of 10mg/100g anesthesia after 30 days of conventional feeding);surgery group(group S,n=20,splenectomy was performed after intraperitoneal propofol injection of 10mg/100g anesthesia after 30 days of conventional feeding);PS intervention group(group PS,n=20,splenectomy was performed after intraperitoneal propofol injection of 10mg/100g anesthesia after 30 days of conventional feeding and 99%phosphatidylserine 50 mg/kg*d gastric administered).Each group performed four times a day,five consecutive days of Morris Water Maze training before the operation.At the 6th day,anesthesia or surgical treatment was performed according to the relevant groups.10 rats were randomly selected on the 3rd day postoperation and the remaining 10 were selected on the 7th day postoperation performed place navigation test and spatial probe test.Then the rats were sacrificed under anesthesia and got the rat hippocampuses.Water content of hippocampus,ATP,SOD,MDA,NO,Na+K+ATPase and Ca2+Mg2+ATPase were tested,electron microscope was used to observe the rat hippocampuses at the 7th day postoperation.Results:Morris Water Maze test showed at the 3rd day postoperation the incubation period of group S was higher than that in group C and group A,while the incubation period of group PS was lower than that in group S and higher than that in group C and group A.At the 7th day postoperation the incubation period of group S was higher than that in group C and group A,while the incubation period of group PS was lower than that in group S.At the 3rd and 7th day postoperation the number of rat platform crossing in group S was lower than that in group C and group A,while the number of rat platform crossing in group PS was higher than that in group S.At the 3rd day postoperation the hippocampal water content in group S was higher than that in group C and group A,while the hippocampal water content in group PS was lower than that in group S.At the 7th day postoperation the hippocampal water content in group S was higher than that in group C and group A.At the 3rd and 7th day postoperation the hippocampal ATP content in group S was lower than that in group C and group A,while the rat hippocampal ATP content in group PS was higher than that in group S and lower than that in group C and group A.At the 3rd day postoperation the hippocampal SOD activity of group S was lower than that in group C and group A,while the SOD activity of group PS was higher than that in group S and lower than that in group C and group A.At the 7th day postoperation the hippocampal SOD activity of group S was lower than that of group C and group A.At the 3rd and 7th day postoperation the hippocampal MDA content in group S was higher than that in group C and group A,while the hippocampal MDA content in group PS was lower than that in group S and higher than that in group C and group A.At the 3rd and 7th day postoperation the hippocampal NO content in group S was higher than that in group C and group A,while the hippocampal NO content in group PS was lower than that in group S and higher than that in group C and group A.At the 3rd and 7th day postoperation the activity of Na+K+ATPase and Ca2+Mg2+ATPase in group S was lower than those in group C and group A,while the activity of Na+K+ATPase and Ca2+Mg2+ATPase in group PS was higher than those in group S and lower than those in group C and the group A.At the 7th day postoperation the electron microscope showed hippocampus in group S cytoplasmic endoplasmic reticulum dilatation,some mitochondria were swollen with vacuoles,synaptic vesicles were dilated,and the chromatin of neuronal nucleus accumulated.Mitochondrial swelling lightened,endoplasmic reticulum dilation and neuronal nuclear chromatin distribution were more uniform in group PS.Conclusion:At the 3rd and 7th day postoperation,the cognitive function of the aged rats undergoing splenectomy were decreased and accompanied by oxidative stress response in hippocampus.Preoperative intervention of phosphatidylserine could partially improve the cognitive function and reduce the degree of oxidative stress response in hippocampus.Part Ⅲ The effect of phosphatidylserine intervention to activation on microglial,blood-brain barrier permeability and apoptosis of hippocampal nerve cells in elderly rats undergoing splenectomyObjective:To investigate whether phosphatidyl serine intervention may reduce apoptosis of hippocampal nerve cells by inhibiting activation on Ml microglial and decreased blood-brain barrier permeability in elderly rats undergoing splenectomy.Materials and methods:80 male aged Sprague-Dawley(SD)rats(CL),weight:380-420g,age:18 months and above.Rats were divided into four groups according to the random principle:control group(group C,n=20,conventional feeding for 30 days);anesthesia group(group A,n=20,resuscitation after intraperitoneal propofol injection of 10mg/100g anesthesia after 30 days of conventional feeding);surgery group(group S,n=20,splenectomy was performed after intraperitoneal propofol injection of 10mg/100g anesthesia after 30 days of conventional feeding);PS intervention group(group PS,n=20,splenectomy was performed after intraperitoneal propofol injection of 10mg/100g anesthesia after 30 days of conventional feeding and 99%phosphatidylserine 50 mg/kg*d gastric administered).In each group 10 rats were randomly selected on the 3rd day postoperation and the remaining 10 were selected on the 7th day postoperation were sacrificed under anesthesia and got the rat hippocampuses.The microglial activation-related inflammatory factors as well as permeability of the blood-brain barrier to Evans blue were tested,westen-blot was used to detect the expression of protein ZO-1、protein occluding and protein claudin,FACS was used to detect the apoptosis of hippocampus neuron.Results:At the 3rd day postoperation the IL-6 level in group S was higher than that in group C and group A,while the IL-6 level in group PS was lower than group S and higher than that in group C and group A.At the 7th day postoperation the IL-6 level in group S was higher than that in group C and group A,while the IL-6 level in group PS was lower than that in group S.At the 3rd day and the 7th day postoperation the IL-1βlevel in group S was higher than that in group C and group A,while the IL-1β level in group PS was lower than that in group S.At the 3rd day postoperation the IL-4 level in group S was lower than that in group C and group A,while the IL-4 level in group PS was higher than that in group S.At the 3rd day and the 7th day postoperation the IL-10 levels in group S was lower than that in group C and group A,while the IL-10 levels in group PS was higher than that in group S.At the 3rd day postoperation the EB exudation in group S was higher than that in group C and group A,while the EB exudation in group PS was lower than that in group S and higher than that in group C and group A.At the 7th day postoperation the EB exudation in group S was higher than that in group C and group A,while the EB exudation in group PS was lower than that in group S.At the 3rd day postoperation the expression of ZO-1 and occludin in group S were lower than those in group C and group A,while the expression of ZO-1 and occludin in group PS were higher than those in group S and lower than those in group C and group A.At the 7th day postoperation the expression of ZO-1 and occludin in group S were lower than those in group C and group A,while the expression of ZO-1 and occludin in group PS were higher than those in group S.At the 3rd day and the 7th day postoperation the percentage of apoptotic hippocampus neuron in group S was higher than that in group C and group A,while the percentage of apoptotic hippocampus neuron in group PS was lower than that in group S and higher than that in group C and group A.Conclusion:At the 3rd day and the 7th day postoperation,M1 microglial activation-related inflammatory factors increased and M2 microglial activation-related inflammatory factors decreased in aged rats hippocampus undergoing splenectomy.,the blood-brain barrier permeability increased,the expression of ZO-1 and occludin decreased and the percentage of apoptotic hippocampus neuron increased.Preoperative intervention of phosphatidylserine may partially increase M2 microglial activation-related inflammatory factors and decrease M1 microglial activation-related inflammatory factors in hippocampus,blood-brain barrier permeability decreased,the expression of ZO-1 and occludin increased and reduce the percentage of apoptotic neurons in the hippocampus at the 3rd day and the 7th day postoperation.