Objective The incidence of Alzheimer's disease(AD)in postmenopausal women is significantly increased with ageing.Menopause induced ovarian estrogen deficiency has been regarded as one of the causative risk factor of AD,but currently the detail mechanisms underlying estrogen regulation on the hippocampus remain unclear.This study aimed to uncover the potential pathways and key molecules through which estrogen deficiency induced by ovariectomy promotes AD pathogenesis by integrating transcriptomic and proteomic analyses.Methods The ovariectomy(OVX)mouse model was established to simulate postmenopausal estrogen decline.Transcriptomic(n=3)and proteomic(n=3)differences in the hippocampal tissues of OVX mice were analyzed using RNA sequencing and protein mass spectrometry.Co-trend molecules identified from both omics datasets were subjected to GO/KEGG enrichment and cluster analyses.These molecules were further compared with the human AD hippocampal database(AlzDate)for expression trend consistency;and online resources such as String,GeneCards/Uniports were employed to analyze protein interactions and networks of the co-trend genes to identify key molecules.Results A total of 139 differentially expressed genes(DEG)and 248 differentially expressed proteins(DEP)were identified,with 18 molecules showing consistent expression trends in both omics.Functional enrichment analysis revealed that these molecules were primarily involved in axonogenesis,cytoskeleton dynamics regulation,and long-chain fatty acid transport.After comparison with the human AD database,11 of the 18 co-trend molecules exhibited expression inhibition consistent with AD-associated genes in the human hippocampal databases.Interaction network analysis identified Aldoc(involved in glycolysis),Aldh6a1(involved in ROS scavenging),Etnppl(involved in mitochondrial energy metabolism),and Itih3(involved in hyaluronic acid-mediated extracellular matrix remodeling)as key genes contributing to AD pathogenesis.Additionally,3 co-trend molecules(Prxl2a,Set,Plin4)were not detected in human AD databases.Conclusion Postmenopausal estrogen deficiency decreases the expression levels of hippocampal Aldoc,Etnppl and Aldh6a1 then affects mitochondrial energy metabolism;and decreases levels of Itih3 expression then disables extracellular matrix remodeling.These effects collectively impair hippocampal axonal regenerative capacity,destabilize the cytoskeleton,and hinder the transport of long-chain fatty acids,ultimately driving the progression of AD pathology.

OBJECTIVE:To establish the method for the determination of mildronate in human plasma and urine,and to study the pharmacokinetic characteristics in healthy volunteers. METHODS:After precipitating plasma and urine sample,LC-MS/MS method was adopted. Dikma Diamonsil C18 column was used with mobile phase consisted of methanol-water(containing 0.2% for-mic acid,0.3% ammonium acetate)(31∶69,V/V)at the flow rate of 0.6 ml/min. ESI was adopted in MRM mode,by using nega-tive ion. The ion for quantitative analysis were m/z 147.10→58.20 (mildronate) and m/z 152.00→110.10 (internal standard,acet-aminophen). The pharmacokinetic parameters of mildronate with single administration and multiple administration were calculated by using DAS 2.1 software and compared. RESULTS:The linear range of mildronate in plasma were 0.02-20 ng/ml(r=0.999 3) and in urine were 0.05-40 ng/ml(r=0.998 2). The lowest limits of quantitation were 0.02 and 0.05 ng/ml. Precision and recovery met the requirements of biological specimen determination,and endogenous impurities hadn’t effect on the determination. The main pharmacokinetics parameters of low-dose,medium-dose and low-dose(250,500,750 mg)of mildronate in plasma with single ad-ministration were as follows:t1/2 were(3.39±0.81),(5.52±0.57)and(5.32±0.96)h;tmax were(0.80±0.45),(1.38±0.43)and (1.10±0.36)h;cmax were(4.17±1.46),(8.08±1.04)and(15.04±1.86)ng/ml;AUC0-36 h were(24.55±5.81),(45.50±7.07)and (85.60 ± 13.09)ng·h/ml. In the dose range,cmax,AUC0-36 h h had a linear relationship with dose (R2 were 0.974 5 and 0.968 3). The main pharmacokinetic parameters of low-dose of mildronate with multiple administration after keeping stable were as follows:cmin was(0.28 ± 0.10)ng/ml;AUCs was(38.78 ± 4.18)ng·h/ml;cs was(1.62 ± 0.17)ng/ml;DF was(3.81 ± 1.14);t1/2 was(6.17 ± 1.46)h;tmax was(1.20 ± 0.33)h;cmax was(6.46 ± 1.96)ng/ml;AUC0-36 h was(40.33 ± 4.65)ng·h/ml;accumulation factor of cmax and AUC were(1.73±0.90)and(1.64±0.40). Compared with single administration,t1/2,cmax and AUC of mildronate with multiple admin-istration after keeping stable all changed,and tmax had no signifi-cant difference. After single administration,26 h accumulative excretion rate of those groups were (0.004 009 ± 0.001 1)%, (0.004 026±0.001 01)% and(0.003 858±0.000 68)% respec-tively. CONCLUSIONS:Established method is sensitive,accurate and specific,and suitable for the determination of mildronate concentration in human plasma and urine and pharmacokinetics study. Mildronate capsule shows certain accumulation effect in healthy volunteers,and linear pharmacokinetic characteristics.