Objective: To analyze differences in the expression levels of lipid metabolism-related proteins in adi- pose tissue exosomes between obese mice and wild-type mice using proteomic techniques . Methods: Wild-type (WT) and obese (ob/ob) model mice of the same age were selected , with 8 mice per group . Adipose tissue from both groups was minced and cultured for 48 hours . Conditioned media was collected , and exosomes were isolated u- sing differential centrifugation . Liquid chromatography-tandem mass spectrometry ( LC-MS/MS) was utilized for proteomic analysis to screen differentially expressed proteins ( DEPs) . Gene ontology ( GO) enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed on the DEPs . Heatmaps were generated to visualize DEPs expression patterns , and Western blot was employed to validate DEPs expression levels . Results: Exosomes were successfully extracted from the culture supernatant of adipose tissues from mice in the WT group and the ob/ob group . Mass spectrometry analysis identified a total of 25 629 peptides and 3 376 proteins . Compared with the WT group , there were 699 proteins with high expression and 632 proteins with low expression in the exosomes derived from adipose tissues of ob/ob mice . Both GO and KEGG analyses showed that DEPs were mainly enriched in metabolic pathways . Heatmap analysis visualized the expression patterns of metabolism-related DEPs , and the expression levels of lipid metabolism-related proteins such as acyl-CoA syn- thetase long-chain family member 1 (ACSL1) , apolipoprotein E (ApoE) , and albumin (Alb) changed significant- ly in the obese state (P < 0. 05) . Western blot verification results showed that the expression of ApoE and Alb pro- teins in the adipose tissue-derived exosomes of ob/ob mice decreased ( P < 0. 01) . Conclusion In the obese state , the expression levels of lipid metabolism-related proteins in mouse adipose tissue exosomes are significantly altered . These differentially expressed proteins may thus serve as potential molecular targets for treating obesity and its associated metabolic complications .

Objective : To construct a knockdown recombinant adeno⁃associated virus (AAV⁃shCTSK) targeting the mouse Cathepsin K(CTSK) gene and assess the knockdown efficiency of AAV⁃shCTSK in mice , and to investigate its impact on lipid storage within adipose tissue. Methods : ShRNA primers specific to both the negative control (NC) and CTSK were designed , annealed , and integrated into the backbone vector. Following clone selection and sequencing for validation , recombinant plasmids were purified. Adeno⁃associated viral vectors , along with packaging and helper plasmids , were co⁃transfected into 293T cells using the transfection reagent PEI for adeno⁃associat⁃ed viral packaging and amplification. The resultant AAV⁃shNC and AAV⁃shCTSK were injected into the epididymal adipose tissue of mice via in situ injection. Two weeks post⁃injection , the expression of CTSK protein was evaluated through immunoblotting assay and the size of intracellular lipid droplets in mouse adipose tissue was detected by HE staining. Results : Successful acquisition of AAV⁃shNC and AAV⁃shCTSK adeno⁃associated viruses was achieved. In mice subjected to in situ injection of AAV⁃shCTSK , effective knockdown of CTSK in adipose tissue was confirmed , accompanied by a significant reduction in the size of white adipocytes. Conclusion A knockdown adeno-associated virus targeting CTSK in mice is successfully constructed , and CTSK knockdown in adipose tissue leads to a notable decrease in lipid content.