L-Carnosine Promotes The Proliferation And Differentiation Of Pig Satellite Cells Via Activating M TOR And Wnt/?-catenin Signaling Pathway

Satellite cells(SCs)play an important role in post-natal muscle growth and regeneration,the purpose of this study was conducted to investigate the effects of L-carnosine on proliferation and differentiation of SCs,revealing the underlying mechanism.Experiment 1:One-day old and healthy landrace pigs were selected to isolated SCs in this study,and then the SCs were purified via differential adhesion method;SCs were seeded in 96-well plates at a density of 1×104 cells/mL in DMEM/F12 medium with 10%FBS(GM)).24 h after seeding,the cultures were treated with fresh GM containing different levels of L-carnosine.The MTT assay showed that supplementation of L-carnosine at dose of 0.2,2,1 and 2 mM/L significantly increased(P<0.05)proliferation,which was significantly inhibited(P<0.05)by 20 mM/L of L-carnosine.Therefore,0,0.2,2 and 20 mM/L were selected for the nest experiments.Experiment 2:SCs at the third generation were seeded in 96/6-well plates.24 h after seeding,the cultures were treated with fresh GM containing different levels of L-carnosine(0,0.2,2 and 20 mM/L).The effects of carnosine on cell proliferation were determined at 24,48,72,96 and 120 h after seeding.The results showed that:supplementation of L-carnosine at dose of 0.2 and 2 mM/L significantly increased(P<0.05)the SCs proliferation at 72,96 and 120 h after seeding,but single or mixed addition of ?-alanine and L-histidine did not significantly(P>0.05)affect SCs proliferation as compared to the control;SCs proliferation,which were significantly decreased by 20 mM/L of L-histidine(P<0.05),were not affected(P>0.05)by 20 mM/L of L-carnosine or ?-alanine.In addition,supplementation of L-carnosine at 2 mM/L significantly decreased(P<0.05)the rate of SCs in G0/G1 phase and the relative expression of Myod gene,while significantly increased(P<0.05)the rate of SCs in S phase and the relative expression of IGF-1 gene.SCs size was not significantly affected(P>0.05)by L-carnosine.Experiment 3:SCs at the third generation were cultured in 96-well plates.24 h after seeding,the cultures were treated with fresh GM containing different levels of L-carnosine(0,and 2 mM/L).The effects of carnosine on m TOR signaling pathway were determined by Western Blot at 96 h after seeding.The results showed that the expressed levels of IGF-1?Akt?m TOR?S6K were significantly increased(P<0.05)by L-carnosine compared with the control.Experiment 4:SCs at the third generation were cultured in 96-well plates.24 h after seeding,the cultures were treated with fresh differentiation medium containing different levels of L-carnosine(0,0.2,2 and 20 mM/L).The effects of carnosine on cell differetiation were investigated.The results showed that supplementation of L-carnosine at 0.2 and 2 mM/L significantly improved the fusion rates of SCs at 24,48,72,96 and 120 h after inducing,as well as the ability of migration of SCs.Western Blot assay indicated that L-carnosine(2 mM/L)significantly increased(P<0.05)the expression of fast-,slow-and total-MyHC,and the Wnt/?-catenin signaling pathway was activated by L-carnosine(P<0.05).