The Protective Effect And Mechanism Of Pioglitazone On Compression-induced Nucleus Pulposus Mesenchymel Stem Cells Apoptosis

Objectives: The present study aimed to investigate the protective effect of pioglitazone on compression-induced nucleus pulposus mesenchymel stem cells(NP-MSCs)apoptosis and further to explore the mechanism by which this process occurs.Methods: Human NP-MSCs were isolated and cultured from patients undergoing lumbar discectomy and were identified by detecting immunophenotypes and multilineage differentiation.Cell viability was analyzed with Cell Counting kit-8(CCK-8).The cytotoxicity of NP-MSCs were detected by LDH assays and live/dead staining.The actin cytoskeletal organization of NP-MSCs were observed by using phalloidin staining.Human NP-MSCs apoptosis was determined by Annexin V/PI double staining and TUNEL assays.Intracellular reactive oxygen species(ROS)was detected by fluorescent probes DCFH-DA,while mitochondrial membrane potential(MMP)was determined by JC-1.The cellular ultrastructure was analyzed by transmission electron microscopy(TEM).Besides,mitochondrial apoptosis-related proteins(cyto.cytochrome c,Bax,Bcl-2,cleaved caspase-9 and cleaved caspase-3)were analyzed by western blot.Results: Our results indicated that NP-MSCs highly expressed surface markers,including CD105,CD73 and CD90,and expressed less CD34 and HLA-DR,having the ability of osteogenesis,adipogenesis and chondrogenic differentiation.These results revealed that the isolated cells satisfied the criteria of MSC stated by the International Society for Cellular Therapy(ISCT).The results of CCK-8 showed that pioglitazone protected the loss of cell viability induced by compression.The LDH assays and live/dead staining revealed that pioglitazone alleviated the toxic effects in compression-induced NP-MSCs.The results of phalloidin demonstrated that the tubular network structures and stress fibers of NP-MSCs were damaged obviously under compression and pioglitazone can reverse the adverse outcomes.The compression-induced apoptosis of human NP-MSCs was reduced when pretreated with pioglitazone according to Annexin V/PI double staining and TUNEL assays.Pioglitazone decreased compression-induced overproduction of ROS and MDA and reversed compression-induced MMP decrease.Ultrastructure collapse of the mitochondria exhibited a nobale improvement by pioglitazone in compression-induced human NP-MSCs according to transmission electron microscopy(TEM).Furthermore,the molecular data showed that pioglitazone significantly decreased the expression of apoptosis-associated proteins,including cyto.cytochrome c,Bax,cleaved caspase-9,cleaved caspase-3 and promoted Bcl-2 expression.Conclusions: These results indicated that pioglitazone protected compression-induced NP-MSCs apoptosis.The molecular mechanism of pioglitazone on compression-induced cell apoptosis in NP-MSCs may be related to mitochondrial pathway.