Microarray data obtained:
Through the research from the Gene Expression Omnibus (GEO) database, the gene expression profile, GSE 70918 (GPL 19271, Affymetrix Rat Gene 2.1 ST Array) was obtained. In this profile, comprising 4 samples, of which 2 were cultivated with PRP and the other 2 samples were cultivated with platelet-poor plasma (PPP). All of the 4 samples were included into this study.
Identification of differently expressed genes (DEGs)
The GEO2R tool was used to analyze the differently expressed genes. And the threshold of the DEGs in our study was set as |logFC|>1 and p-value < 0.05.
(Protein-protein interaction) PPI network construction and module selection
The PPI network based on commonly differentially expressed genes was constructed by Search Tool for the Retrieval Interacting Genes (STRING) database. The Cytoscape (version 3.8.0) was used to analyze and visualize the correlations among genes. Molecular Complex Detection (MCODE; version 1.31) was used to determine the top 3 correlated gene modules.
KEGG pathway enrichment analysis construction
The Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.8) was used to analyze the KEGG functional enrichment based on the DEGs.
Cell viability evaluated by MTT test
The passage-3 ADSCs were digested and diluted, and the mixture was transferred to a 96-well culture plate at 100 µL cell suspension per well and incubated at 37 ℃, in a 5% CO2-saturated humidity float tank for 24 hours. 5-Aza was then added to each well containing ADSCs at concentrations of 0, 10, 20, 30, 40 µmol/L. ADSCs group were labeled groups A, B, C, D and E. Following incubation under the same conditions as before for 24 hours, we added 50 µL MTT solution to each well and incubated again under the same conditions for another 4 hours, terminating the reaction of each well by aspirating the culture medium. To each well was added 150 µL DMSO solution after terminating the 4 hours of incubation, with the plate placed on the table concentrator. Finally, the absorbance of each well was measured at 550 nm (OD) to detect the cell viability under the induction of different concentrations of 5- Aza.
Aging-associated β-galactosidase (SA- β-gal) staining
According to the instructions of the reagent, the aging β-galactosidase staining kit (CST Company) was used to detect the activity of SA- β-gal in ADSCs. SA- β-gal positive cells were quantified as a percentage of the total number of cells analyzed.
Immunohistochemical staining and Semi-quantitative analysis
First, we digested and diluted the cells from each group 9 days after induction; then we added 1 mL of the solution to each well of the 12-well culture plate that had a cell slide placed in every well in advance. We next extracted the slides from each well after culturing at 37 ℃ in a 5% CO2-saturated humidity incubator for 24 hours. We washed the slides once with 300 µL phosphate buffer solution (PBS) and did not remove the liquid for 5 minutes until the cells were saturated. Next, we added 150 µL 4% paraformaldehyde fixative to every slide and left them undisturbed for 30 minutes before adding 150 µL 0.1% Triton x-100 to each and left at room temperature for 3 minutes. We washed the slide 3 times with 300 µL PBS, each washing process lasting 5 minutes. The ensuing step was to drop 200 µL 5% blocking solution (BSA) to each slide and incubate in a 5% CO2 incubator for 1 hour. Each slide was incubated overnight at 4 ℃ after being mixed with 150 µL α-SMA (1:200) diluted with 1% BSA. The slides were washed with 300 µL PBS 5 times the following day, each washing process lasting 5 minutes. 150 µK of the secondary antibody (1:200) diluted with 1% BSA was then added to each slide after the slides were blocked at room temperature with 200 µK 1% BSA for 15 minutes. Next, we washed the slides 5 times with 300 µL PBS for 5 minutes each time. 150 µL Hoechst33258 stain (C1011 Beyotine, China) was added to each slide in a dark environment and incubated for 30 minutes at room temperature. Finally, we observed the cells under a fluorescence microscope, photographed, and stored them.
Intracellular total RNA extraction: Pancreatic enzymes were used to digest the cells that were collected after centrifuging the liquid in the flask. Next, equal volumes of Trizol lysate were added to enable the collected cells to split and decompose. The schizolytic cells were then transferred into another tube without RNA enzymes, and 200 µL pre-cooling chloroform was added per milliliter of Trizol, and the mixture was centrifuged for 15 minutes. The supernatant was absorbed after centrifugation and transferred into another tube without RNA enzymes, where an identical volume of isopropanol was added to the tube, followed by another centrifugation. This later centrifugation yielded RNA sediments that were preserved in a -20 ℃ surrounding for 30 minutes. The sediments were washed with 75% ethyl alcohol and centrifuged for 5 minutes, and the supernatant was discarded after washing and centrifuging the sediments twice. cDNA synthesis: The reverse transcription system was prepared using a reverse transcription kit according to instructions provided in the protocol of the kit.
After washing with PBS for 2 - 3 times, it was added to M16 culture medium preheated to 37 ℃ and containing 400 nmol/L Mito-Tracker Red. The cells were stained at 37 ℃ for 20 minutes. After staining, the cells of each group were washed with PBS for 2 - 3 times, and the cells of each group were placed on the glass slides, covered with glass slides, and observed and photographed under the fluorescence microscope.
Transmission electron microscope
The cells of each group cultured for 72 hours were fixed with 2.5% glutaraldehyde and 1% osmium acid, dehydrated with acetone, prepared, lead and uranium were re-stained with 10 minutes, and the autophagosomes were observed and photographed under transmission electron microscope.
Graphpad 8.0.2 and R 4.0.2 were used to perform statistical analysis. Expressed data were shown as mean ± SD. Student’s t test was used to evaluate the statistical significance of different 3 groups. The p value less than 0.05 was considered as significant.