Adaptive Survival Of Bone Marrow Mesenchymal Stem Cells After Starvation Induction And The Effect Of Its Secreting Extracellular Vesicles On Co-cultured Chondrocytes

Objective To observe the changes of rat bone marrow mesenchymal stem cells(BMSCs)after starvation induction,and to study the effect of starving BMSCs and their secreted extracellular vesicles on co-cultured chondrocytes under serum and glucose deprivation,and to explore the mechanism preliminarily.Methods 1.The BMSCs of rat were isolated by the whole bone marrow adherence,and the stem cell surface markers were detected by flow cytometry;adipogenic induction medium,osteogenic induction medium and chondrogenic induction medium were used to culture BMSCs.2.The chondrocytes of rat were extracted and identified by type II collagen immunofluorescence staining.3.The BMSCs were cultured in serum and glucose deprivation medium,and the morphological of BMSCs were observed by electron microscope.The internal changes of BMSCs after starvation were observed by TEM.4.The chondrocytes were cultured under starving conditions,and the apoptosis of chondrocytes after co-culture with starving BMSCs was detected by flow cytometry.The apoptosis-related proteins Bax,Bcl-2,and cleaved-caspase-3were detected by western blot.5.Transfect m RFP-GFP-LC3 adenovirus into chondrocytes,observed the autophagy of chondrocytes after co-culture with starving BMSCs,and detected the expression of autophagy-related protein LC3 II by Western blot.6.Isolation of starving BMSCs-EVs was performed using an ultracentrifugation method.The morphology of vesicles was observed by TEM,NTA determined the size distribution of vesicles,and the characteristic proteins of vesicles were detected by Western blot.The endocytosis of starving BMSCs-EVs by chondrocytes was observed by fluorescence microscope;7.The apoptosis of chondrocytes under starving conditions was detected by flow cytometry and observed the effect of starving BMSCs-EVs on cartilage apoptosis.autophagy and apoptosis-related proteins were detected by Western blot.8.Using mi RNA sequencing technology detected High-expressed mi RNAs in starving BMSCs-EVs,and the corresponding target genes were predicted,and KEGG and GO enrichment analysis were performed.Results 1.The BMSCs of rat were successfully isolated,and it had the ability to differentiate into bone,adipogenic and cartilage.2.Chondrocytes could be successfully isolated by collagenase type II digestion.The p3 generation chondrocytes had high purity and strong proliferative ability,which could meet the requirements of subsequent experiments.3.No obvious death of BMSCs after 72 h starvation induction.TEM results showed that starving BMSCs to secrete more extracellular vesicles and increase mitochondria.4.Serum and glucose deprivation culture of chondrocytes could induce chondrocytes apoptosis.However,adding starving BMSCs to the culture system could reduce chondrocytes apoptosis;Western blot demonstrated that the expression of anti-apoptotic proteins in chondrocytes increased after co-culture with starving BMSCs,and the expression of pro-apoptotic proteins decreased.5.The autophagy of chondrocytes could be induced under serum and glucose deprivation culturing,and adding starving BMSCs to the culture system could reduce the autophagy of chondrocytes.Western blot demonstrated that the expression of autophagy-related protein LC3 II was increased in chondrocytes after co-culture with starving BMSCs.6.TEM representative images,NTA detection results and marker protein detection proved that the starving BMSCs-EVs were successfully extracted.7.Starving BMSCs-EVs could also reduce chondrocytes apoptosis under serum and glucose deprivation conditions,and starving BMSCs-EVs played a similar function to the autophagy inhibitor 3MA.Starving BMSCs-EVs inhibited autophagy and attenuated apoptosis of chondrocytes.8.The results of transcriptome sequencing showed that starving BMSCs-EVs were enriched in a large number of mi RNAs,and the first 50 mi RNAs were subjected to functional enrichment analysis.KEGG pathway enrichment indicated ‘MAPK signalling pathway’,‘focal adhesion’,‘PI3K-Akt signalling pathway’ and ‘apoptosis’ as enriched pathways.GO analysis revealed that ‘positive regulation of transcription from RNA polymerase II promoter’and ‘positive regulation of cell proliferation’ in the biological process category;‘nucleus’ and ‘cytoplasm’ in the cellular component category and ‘protein binding’and ‘DNA binding’ in the molecular function category were significant.These results showed that mi RNAs from starving BMSCs-EVs may contribute to the anti-apoptosis of chondrocytes under starving condition.Conclusion 1.Serum and glucose deprivation culture did not cause massive death of BMSCs,and BMSCs showed adaptive survival over time and secreted more vesicles.2.In vitro,serum and glucose deprivation culture could induce chondrocyte apoptosis,but adding starving BMSCs could inhibit chondrocyte autophagy and reduce chondrocyte apoptosis.3.Starving BMSCs-EVs could also regulate chondrocyte autophagy and reduce chondrocyte apoptosis in starvation environment.4.Starving BMSCs/BMSCs-EVs assisted the anti-apoptosis of chondrocytes due to the starving BMSCs-EVs carrying a large number of mi RNAs that can regulate apoptosis.