The Mechanism Of Mesenchymal Stem Cell-derived Exosome Inhibits High Glucose-induced Fibroblasts Transdifferentiation

Objective: Recent studies have shown that the development of diabetic cardiomyopathy is closely related to the cardiac fibrosis,which is associated with the transdifferentiation of fibroblasts into myofibroblasts and excessive deposition of collagen.The aim of this study is to explore the mechanism of high glucose-induced fibroblasts transdifferentiation by culturing fibroblasts under high glucose(25 mmol/L)condition to mimic the high glucose environment in vivo and to study whether mesenchymal stem cell-derived exosome(MSC-Exo)can inhibit high glucose-induced fibroblasts transdifferentiation.Our research will pave the way to the development of novel treatment for diabetic cardiomyopathy.Methods: High glucose induced transdifferentiation of human skin fibroblasts cell line(BJ cells)was chosen as an in vitro model to study mechanism of diabetes induced cardiac fibrosis.1.To establish a model of fibroblasts transdifferentiation into myofibroblasts,the BJ cells were exposed to high glucose(25 mmol/L)for 24 h and 48 h,then the protein and mRNA expression of ?-SMA were determined;2.In order to examine whether high glucose can promote the activation of Smad2/3 and whether SB525334 can inhibit high glucose induced-fibroblasts transdifferentiation,the BJ cells were pretreated with SB525334(10 ?mol/L)prior to high glucose treatment and the expression of ?-SMA was examined by Western Blot;3.The expression of collagen? in BJ cells exposed to high glucose was examined by Western Blot;4.The effect of high glucose on BJ cells proliferation was detected by EdU proliferation kit;5.BJ cells were transfected with TGF-?1 siRNA by Lipofectamine 2000 to study whether endogenous TGF-?1 knock down can inhibit the BJ cells transdifferentiation induced by high glucose;6.When the BJ cells were exposed to high glucose,MSC-Exo was added at the same time,to study whether MSC-Exo can inhibit high glucose-induced BJ cells transdifferentiation;7.The expression of TNF-? and IGF-1 in BJ cells were detected by ELISA.Results: 1.The expression of ?-SMA was detected by Western Blot and qPCR after high glucose treatment for 24 h and 48 h.The results showed that the levels of ?-SMA protein and mRNA were significantly increased in BJ cells treated with high glucose,which suggests that the fibroblasts have transdifferentiated into myofibroblasts;2.BJ cells were pretreated with SB525334 for 30 min and then were stimulated with high glucose.We found that SB525334 inhibited the increase of ?-SMA expression induced by high glucose.We also found that high glucose can promote Smad2/3 activation,which can be inhibited by SB525334 pretreatment;3.The expression of collagen? was significantly up-regulated when BJ cells were treated with high glucose,and this process was inhibited by SB525334;4.High glucose promoted the proliferation of BJ cells,and SB525334 also can inhibit this process;5.The transdifferentiation was significantly inhibited by silencing the endogenous TGF-?1 with siRNA in BJ cells;6.MSC-Exo can inhibit high glucose-induced BJ cells transdifferentiation via Smad2/3 signaling pathway;7.High glucose did not alter the expression of TNF-? and IGF-1 in BJ cells,which indicated that TNF-? and IGF-1 were not involved in high glucose-induced fibroblasts transdifferentiation.Conclusions: High glucose induced the transdifferentiation of BJ cells into myofibroblasts through the activation of TGF-?1/Smad2/3 signaling pathway,and increased the expression of collagen?,and promoted the proliferation of BJ cells;MSC-Exo inhibited high glucose-induced BJ cells transdifferentiation via TGF-?1/Smad2/3 signaling pathway.However,TNF-? and IGF-1 were not involved in the transdifferentiation.