Astaxanthin Promotes the Proliferation and Differentiation of Chicken Muscle Stem Cells via AMPK/mTOR Signaling Pathway
[Objective]To study the effect of astaxanthin(AST)on the proliferation and differentiation of chicken muscle stem cells(Ch-MuSCs)and its potential mechanism.[Method]Primary Ch-MuSCs were isolated and treated with different concentrations of AST(0,0.3,0.6,1.25,2.5,and 5 μmol/L)for 24 h.Cell viability and proliferation were analyzed using 7-AAD and Calcein AM staining,MTT assay and EdU assay.Myotube development was evaluated through immunofluorescence staining.Western blot analysis was performed to examine the expression of key proteins in the AMPK/mTOR signaling pathway,including PI3k,AKT,phosphorylated PI3k,phosphorylated AKT,mTOR,AMPK,phosphorylated mTOR,and phosphorylated AMPK.[Result]Both 0.6 and 1.25 μmol/L AST treatments significantly increased cell activity and reduced necrotic cells(P＜0.01),with the 1.25 μmol/L AST treatment exhibiting the most significant effect on cell proliferation(P＜0.01).Immunofluorescence staining of Titin and MyoD revealed that treatment with 1.25 μmol/L AST significantly increased the length of Titin-positive muscle tubes(P＜0.05)and the number of MyoD-positive nuclei per muscle tube(P＜0.05).Western blot demonstrated that the levels of p-PI3K and p-AKT were significantly elevated in the 1.25 μmol/L AST treatment group(P＜0.05).The expressions of AMPK and p-AMPK proteins increased,while the expression of mTOR protein decreased(P＜0.05).After adding the mTOR-selective rapamycin,the expressions of AMPK protein and mTOR protein significantly decreased(P＜0.05),but the expression of p-AMPK protein did not change significantly.If AST was used simultaneously,it increased the expressions of AMPK and P-AMPK proteins(P＜0.05),but the expressions of mTOR protein and p-mTOR significantly decreased(P＜0.05).[Conclusion]AST can promote the proliferation and differentiation of Ch-MuSCs by activating AMPK and its upstream signaling molecules PI3K and Akt in the AMPK/mTOR signaling pathway,and regulate the growth rate of Ch-MuSCs by inhibiting mTOR.
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